Complexes of rna  and cationic peptides for transfection and for immunostimulation

ABSTRACT

The present invention relates to a complexed RNA, comprising at least one RNA complexed with one or more oligopeptides, wherein the oligopeptide, which has the function of cell-penetrating peptide (CPP), has a length of 8 to 15 amino acids and has the empirical formula (Arg) l ;(Lys) m ;(His) n ;(Om) o ;(Xaa) x  with the majority of residues being selected from Arg, Lys, His, Om. The invention further relates to a method for transfecting a cell or an organism, thereby applying the inventive complexed RNA. Additionally, pharmaceutical compositions and kits comprising the inventive complexed RNA, as well as the use of the inventive complexed RNA for transfecting a cell, tissue or an organism and/or for modulating, preferably inducing or enhancing, an immune response are disclosed herein.

The present invention relates to a complexed RNA, comprising at leastone RNA (molecule) complexed with one or more oligopeptides, wherein theoligopeptide has a length of 8 to 15 amino acids and has the formula(Arg)_(l)(Lys)_(m)(His)_(n)(Orn)_(o)(Xaa)_(x). The invention furtherrelates to a method for transfecting a cell or an organism, therebyapplying the inventive complexed RNA. Additionally, pharmaceuticalcompositions and kits comprising the inventive complexed RNA, as well asthe use of the inventive complexed RNA for transfecting a cell, tissueor an organism and/or for modulating, preferably inducing or enhancing,an immune response are disclosed herein.

Transfection of nucleic acids into cells or tissues of patients bymethods of gene transfer is a central method of molecular medicine andplays a critical role in therapy and prevention of numerous diseases.Methods for transfection of nucleic acids may lead to immune stimulationof the tissue or organism. Alternatively or additionally, transfectionof nucleic acids may be followed by processing of the information codedby the nucleic acids introduced, i.e. translation of desiredpolypeptides or proteins. DNA or RNA as nucleic acids form alternativeapproaches to gene therapy. Transfection of nucleic acids may also leadto modulation, e.g. suppression or enhancement of gene expression,dependent on the type of nucleic acid transfected. Transfection of thesenucleic acids is typically carried out by using methods of genetransfer.

Methods of gene transfer into cells or tissues have been intensivelystudied in the last decades, however in part with limited success. Wellknown methods include physical or physico-chemical methods such as(direct) injection of (naked) nucleic acids or biolistic gene transfer.Biolistic gene transfer (also known as biolistic particle bombardment)is a method developed at Cornell University, that allows introducinggenetic material into tissues or culture cells. Biolistic gene transferis typically accomplished by surface coating metal particles, such asgold or silver particles, and shooting these metal particles, comprisingthe adsorbed DNA, into cells by using a gene gun. However, biolisticgene transfer methods have not yet been shown to work with RNA, probablydue to its fast degradation. Furthermore, these methods are not suitablefor in vivo applications, a matter which represents a severe practicallimitation.

An alternative physical or physico-chemical method includes the methodof in vitro electroporation. In vitro electroporation is based on theuse of high-voltage current to make cell membranes permeable to allowthe introduction of new DNA or RNA into the cell. Therefore, cell wallsare typically weakened prior to transfection either by using chemicalsor by a careful process of freezing to make them “electrocompetent”. Ifelectrocompetent bacteria or cells (e.g. eukaryotic cells) and DNA (orRNA) are mixed together, the plasmid can be transferred into the cell byusing an electric discharge to carry the DNA (or RNA) into cells in thepath of the spark crossing the reaction chamber.

Another alternative physical or physico-chemical method includes use ofnanoplexes (nanoparticular systems), lipoplexes (liposomal systems), orthe use of polyplexes or cationic polymers. Such nanoplexes(nanoparticular systems) involve use of polyacrylates, polyamides,polystyrene, cyanoacrylates, polylactat (PLA), poly(lactic-co-glycolicacid) (PLGA), polyethyl, etc., as carrier systems for the transport ofnucleic acids into cells or tissues. Lipoplexes or liposomal systemstypically involve use of cationic lipids, which are capable to mimick acell membrane. Thereby, the positively charged moiety of the lipidsinteracts with the negatively charged moiety of the nucleic acids andthus enables fusion with the cell membrane. Lipoplexes or liposomalsystems include e.g. DOTMA, DOPE, DOSPA, DOTAP, DC-Chol, EDMPC, etc.Polyplexes (cationic polymers) typically form a complex with negativelycharged nucleic acids leading to a condensation of nucleic acids andprotecting these nucleic acids against degradation. Transport into cellsusing polyplexes (cationic polymers) typically occurs via receptormediated endocytosis. Thereby, the DNA is coupled to a distinctmolecule, such as Transferrin, via e.g. the polyplex poly-L-lysine(PLL), which binds to a surface receptor and triggers endocytosis.Polyplexes (cationic polymers) include e.g. poly-L-lysine (PLL),chitosan, polyethylenimine (PEI), polydimethylaminoethylmethacrylate(PD-MAEMA), polyamidoamine (PAMAM).

Other well known physical or physico-chemical methods of gene transferinto cells or organisms include methods such as virus based transfectionmethods. As a particular example, DNA viruses may be used as DNAvehicles. Because of their infection properties, such viruses have avery high transfection rate. The viruses typically used are geneticallymodified in a way, that no functional infectious particles are formed inthe transfected cell. In spite of this safety precaution, however, arisk of uncontrolled propagation of the therapeutically active genesintroduced and the viral genes cannot be ruled out e.g. because ofpossible recombination events.

More advantageous in this context is the use of so called translocatoryproteins or of protein transduction domains (PTDs) for the transport ofmacromolecules into cells or tissues. Translocatory proteins areconsidered as a group of peptides capable of effecting transport ofmacromolecules between cells (translocatory proteins), such as HIV tat(HIV), antennapedia (Drosophila antennapedia), HSV VP22 (Herpessimplex), FGF or lactoferrin, etc. In contrast, protein transductiondomains (PTDs) are considered as a group of peptides capable ofdirecting proteins and peptides covalently bound to these sequences intoa cell via the cell membrane (Leifert and Whitton: Translocatoryproteins and protein transduction domains: a critical analysis of theirbiological effects and the underlying mechanisms. Molecular Therapy Vol.8 No. 1 2003). Common to translocatory proteins as well as to PTDs is abasic region, which is regarded as mainly responsible for transport ofthe fusion peptides since it is capable of binding polyanions such asnucleic acids. Without being bound thereto, PTDs may act similar tocationic transfection reagents using receptor dependent non-saturatableadsorptive endocytosis. PTDs are typically coupled to proteins orpeptides in order to effect or enhance a CTL response when administeringa peptide based vaccine (see review: Melikov and Chernomordik,Arginine-rich cell penetrating peptides: from endosomal uptake tonuclear delivery, Cell. Mol. Life Sci. 2005).

Protein transduction domains (PTDs) are sometimes also termed “cellpenetrating peptides” (CPPs) due to their capability of penetrating thecell membrane and thus to effect the transport of (macro-) moleculesinto cells. CPPs are small peptides and typically comprise a highcontent of basic amino acids and exhibit a length of 7 to 30 aminoacids. Macromolecules, which have been shown to be transported intocells via CPPs, include peptides as well as DNA, siRNA or PNAs (peptidenucleic acids), wherein the CPPs are typically bonded to thesemacromolecules via a covalent bond and transfected into the cells.Although cell penetrating peptides (CPPs) have been successfully used tomediate intracellular delivery of a wide variety of molecules ofpharmacological interest both in vitro and in vivo, the mechanisms bywhich cellular uptake occurs still remains unclear. The group of CPPs ishighly diverse and consists of amphipathic, helical peptides such astransportan, penetratin, hydrophobic peptides such as MTS, VP22, MAP,KALA, PpTG20, prolin-rich peptides, MPG-peptides, Pep-1, L-oligomers,calcitonin-peptides, or cationic, hydrophilic arginine-rich peptides,including arginine-rich CPPs, which mediate cellular uptake of(covalently) conjugated molecules via binding to proteoglycanes of thecell, such as the transduction domain of the HIV-1 Tat protein (Review:Deshayes et al. Cell-penetrating peptides: tools for intracellulardelivery of therapeutics. Cell. Mol. Life Sci. 2005). Particularly,arginine-rich CPPs are described as vehicles for proteins or DNA, e.g.plasmid DNA, etc. into cells. Poly-arginines may also be used for thetransport of (macro-) molecules into cells, which typically comprises alength of at least 60 to 80 amino acids (in particular arginines), moretypically from 1000 to 15000 amino acids, and thus represents a highmolecular mass compound. Even though the cellular uptake mechanism forCPPs in general remains unclear, endocytosis is suggested as an uptakemechanism for poly-arginine. Endocytosis is a cellular process by whichmacromolecules may enter a cell without passing through the cellmembrane, wherein three different endocytotic mechanisms have beensuggested (chlathrin-dependent endocytosis, caveolin-dependentendocytosis and/or F actin-dependent endocytosis, see e.g. review:Melikov and Chernomordik, Arginine-rich cell penetrating peptides: fromendosomal uptake to nuclear delivery, Cell. Mol. Life Sci. 2005).Without being bound to any theory, during endocytosis the CPP-complexedmacromolecule first binds to the negatively charged cell surfaceglycosaminoglycans (GAGs), including heparans (HS). Then, the CPP-boundmacromolecule enters cell by chlathrin-dependent endocytosis,caveolin-dependent endocytosis and/or F actin-dependent endocytosis,e.g. by folding of the membrane around the CPP-bound macromoleculeoutside the cell. This results in the formation of a saclike vesicleinto which the CPP-bound macromolecule is incorporated. Trafficking ofthe CPP-bound macromolecule through late endosomes and/or Golgi and/orendoplasmic reticulum (ER) delivers the CPP-bound macromolecule into thecytoplasm, wherein this stage may involve CPP-induced opening of thetransient pores in the lipid bilayer. Alternatively, the CPP-complexedmacromolecule may be transported to other locations in the cell, e.g.into the endosom, dependent on the mode of action required for thespecific purpose. As an example, TLR-7 and TLR-8 receptors are locatedin the endosome. Thus, transfection of cells with immunostimulatory RNA,which may e.g. be ligands of Toll-like receptors (TLRs) selected fromligands of TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3, TLR4,TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13) may lead totransport to the endosomes and (depeding on the specific interaction andthe interaction partners) to e.g. immunostimulation by the RNA ligand.

Cell penetrating peptides (CPPs) as defined above are well known in theart and widely discussed. However, the use of these CPPs (as carriers)is established for the transport of peptides, proteins and DNA as cargo,wherein the CPPs are typically linked to cargo molecules in a covalentmanner. In contrast, cellular transport of RNAs using CPPs was onlyshown for a very limited number of cases, particularly for short RNAsequences, e.g. double stranded siRNA sequences.

By way of example, Futaki et at (The Journal of Biological Chemistry,Vol 276, No. 8, pp. 5836-5840, 2001) disclose the use of carrieroligopeptides (Arg)_(n) having a length of 4-16 amino acids for in vitrotransfer of cargo peptides, wherein the carrier peptides are covalentlylinked to the cargo peptides. A translocation optimum was demonstratedfor (Arg)_(n) having a length of 6 or 8 arginines, respectively.

Trans-membrane transport peptides or peptidomimetics by CPPs was alsoshown by Deshayes et al. (2005, supra). Deshayes et at (2005, supra)disclose the use of oligopeptides Arg₇ and Arg₉ for in vitro transfer ofcargo peptides and in vivo transfer of cargo proteins such ascyclosporin or catalase.

For transfection of cells with macromolecules, such as DNA, peptides orproteins, high molecular weight polypeptides such as poly-L-arginines(e.g. typically having a MW of about 5000 Da to 15 kDa) orpoly-L-lysines (e.g. typically having a MW of about 54 kDa) as well ashigh molecular weight PEI (polyethyleneimin) (e.g. typically having a MWof about kDa) were used according to the art (see also Bettinger et at,Nucleic Acids Research, Vol. 29 No. 18 (2001)). However, high molecularweight poly-L-lysine and PEI appeared to be ineffective as carriermolecules. Further, when using high molecular weight poly-L-arginines athigh concentrations, toxic effects were observed which lead toactivation of the complement system. Thus, efforts were undertaken todevelop low molecular weight transfection agents, such as, e.g., lowmolecular weight poly-arginines. However, such low molecular weightpoly-arginines typically exhibit a low stability of thecarrier-cargo-complex, i.e. the complex formed of, e.g., a poly-arginineas carrier and a DNA molecule as a cargo. Thus, McKenzie et al.(McKenzie et al. A potent new class of reductively activated peptidegene delivery agents; The Journal of Biological Chemistry Vol. 274 No.14, 2000) tried to increase stability of peptide-DNA-complexes bycrosslinking these peptides via glutaraldehyde to the DNA, therebyforming a Schiff's base. However, such crosslinking results in extremelyslow dissociation of the complex in the cell and, consequently,expression of the encoded protein is extremely low over time. In orderto circumvent this problem, McKenzie et al (2000, supra) introducedcysteine residues into the CPP carrier, which stabilize the complex byforming disulfide bonds between CPP and DNA. Upon transfection, thesedisulfide bonds are cleaved in the cell due to the reducing conditionsinside the cell, resulting in increased expression of the encodedpeptides. However, such crosslinking is elaborative and may causefurther undesired modifications of the DNA.

Furthermore, low-molecular weight PEI (e.g. typically having a MW ofabout 2000 Da) and low-molecular weight poly-L-lysines (e.g. typicallyhaving a MW of about 3400 Da) may be used for transfection of suchmacromolecules as mentioned above. However, even though an improvedtransfection was observed for low-molecular weight PEI or poly-L-lysinesin these experiments, expression was not detectable due to formation ofextremely stable complexes of these carrier molecules with the DNA. As aresult, these carrier molecules do not appear to exhibit dissociation oftheir complexed DNA, a necessary step for translation and expression ofthe encoded protein (see Bettinger et al., (2001), supra).

Transport of DNA by CPPs was further shown by Niidome et al (The journalof Biological Chemistry, Vol 272, No. 24, pp. 15307-15312, 1997).Niidome et al (1997, supra) disclose the use of CPPs, particularly ofcationic alpha-helical peptides with a defined arginine content of 25%and a length of 12 or 24 amino acids, respectively, for the transport ofplasmid DNA as cargo moiety. As a result, it was found that long and/orhydrophobic peptides can strongly bind to the DNA and effect transportof DNA into cells. Moreover, Niidome et at (Bioconjugate Chem. 1999, 10,773-780) showed that peptides having a length of 16 to 17 amino acidswere most efficient for the transport of plasmid DNA. However, whenusing small peptides (e.g. of about 12 amino acids) as CPPs,transfection efficiency of DNA into cells turned out to decreasesignificantly.

In order to enhance cellular transfection efficiency of short argininemolecules, Futaki et al. (Bioconjugate Chem. 2001, 12, 1005-1011) usedstearylated oligopeptides (Arg) having a length of 4-16 amino acids.These oligopeptides were used in transfection experiments in comparisonto non-stearylated oligopeptides (Arg)_(n) having a length of 4-16 aminoacids and poly-arginine (MW 5000-15000) for in vitro transfer of plasmidDNA coding for luciferase. Accordingly, carrier peptides used fortransfection were mixed with plasmid DNA and formed a carrier/cargocomplex. A translocation optimum was demonstrated for stearylated(Arg)_(n) having a length of 8 arginines, whereas arginines having alength of 6-7 and 9-15 arginines showed a significantly reduced cellulartransport activity. Furthermore, transport activity of non-stearylatedarginines and poly-arginine exhibited poor results, indicating loss oftransport activity when using these carrier peptides. The observeddifference of transfection efficiency shown by Futaki et al. (2001,supra) for stearylated and non-stearylated carrier peptides is thus dueto the presence of lipid moieties, which significantly change thechemical properties of the CPPs used in these experiments.

According to Kim et al. (Kim et al., Basic peptide system for efficientdelivery of foreign genes, Biochimica et Biophysica Acta 1640 (2003)129-136), short arginine carrier peptides such as (Arg)₉ to (Arg)₁₅ maybe used for complexation and cellular transfection of DNA, encodinggreen fluorescent protein PEGFP-N3. When using arginines (Arg)₉ to(Arg)₁₅, optimum results were obtained with (Arg)₁₅ showing increasingcellular transfection efficiency from (Arg)₉ to (Arg)₁₅. These resultsindicate that optimum transport properties for transfecting cells withDNA may be achieved with an (Arg)_(n) carrier peptide, wherein n is farbeyond 15. However, applicability of short arginine peptides fortransfection purposes was exclusively documented for DNA molecules ascargo moiety by Kim et al. (2003, supra).

Cells may also be transfected by using CPPs in combination with RNA.However, only a small number of working examples were carried out forthe cellular transport of RNA, probably due to its fast degradation andlow stability in complexes. Thus, transfection of RNA using CPPs appearsto be restricted to more stable double stranded RNAs, such as siRNA. Byway of example, Tönges et al. (RNA (2006), 12:1431-1438) usedstearylated octa-arginine (Arg)₈ for the in vitro transfer of doublestranded short siRNA into neuronal hippocampus cells, wherein thestearylated octa-arginine (Arg)₈ forms a complex with siRNA. Based onthe results of Tönges et al. (2006, supra) the stearyl component of thecarrier peptides seems to be indispensible for the transport of siRNA orthe transport of other RNA molecules.

Veldhoen et al. (2006) also published the use of specific CPPs in anon-covalent complex for cellular transfection of double stranded shortsiRNA sequences (Veldhoen et al., Cellular delivery of small interferingRNA by a non-covalently attached cell penetrating peptide: quantitativeanalysis of uptake and biological effect. Nucleic Acids Research 2006).Peptides used by Veldhoen et at (2006) were MPGalpha(Ac-GALFLAFLAAALSLMGLWSQPKKKRKV-Cya) and MPGalpha-mNLS(Ac-GALFLAFLAAALSLMGLWSQPKSKRKV-Cya). These specific peptides wereadditionally modified with an acetyl moiety (Ac) at the N-terminus and acysteamide moiety at the C-terminus. Veldhoen et at (2006) were able toshow transfer of double-stranded siRNA, having a length of about 18 to40 nucleotides, into cells by using the afore-mentioned carrierpeptides.

Summarizing the above, use of CPPs or other carrier peptides for thecellular transport of macromolecules was basically shown for peptidesand for DNA molecules. Few very specific publications disclose cellpenetrating properties of double stranded siRNA.

RNA transfer represents an important tool in modern molecular medicineand exhibits superior properties over DNA cell transfection, since DNAmolecules may lead to serious problems. E.g. application of DNAmolecules bears the risk that the DNA integrates into the host genome.Integration of foreign DNA into the host genome can have an influence onexpression of the host genes and possibly triggers expression of anoncogene or destruction of a tumor suppressor gene. A gene—and thereforethe gene product—which is essential to the host may also be inactivatedby integration of the foreign DNA into the coding region of this gene.There is a particular danger if integration of the DNA takes place intoa gene which is involved in regulation of cell growth. In this case, thehost cell may enter into a degenerated state and lead to cancer or tumorformation. Such undesired integration into the DNA may be even moreproblematic, if the DNA transfected into the cell comprises a potentpromoter, such as the viral CMV promoter. Integration of such promotersinto the genome of the treated cell can lead to undesirable changes inthe regulation of gene expression in the cell. A further disadvantage isthat the DNA molecules remain in the cell nucleus for a long time,either as an episome or, as mentioned, integrated into the host genome.This phenomenon leads both to production of transgenic protein which isnot limited or cannot be limited in time and to danger of associatedtolerance towards this transgenic protein. The development of anti-DNAantibodies (Gilkeson et al., J Clin Invest 95, 1398-1402 (1995)) and theinduction of autoimmune diseases can furthermore be triggered byinjection of DNA. All these risks listed are associated with applicationof DNA. In contrast, they do not occur if RNA, particularly mRNA, isused instead of DNA. For example, mRNA does not integrate into the hostgenome, no viral sequences, such as promoters etc., are required foreffective transcription etc. A disadvantage resulting from the use ofRNA may be due to its instability as compared to DNA (RNA-degradingenzymes, so-called RNases (ribonucleases), in particular, but alsonumerous other processes which destabilize RNA are responsible for theinstability of RNA). However, methods for stabilizing RNA have meanwhilebeen disclosed in the art, such as, for example, in WO 03/051401, WO02/098443, WO 99/14346, EP-A-1083232, U.S. Pat. No. 5,580,859 and U.S.Pat. No. 6,214,804. Methods have also been developed for protecting RNAagainst degradation by ribonucleases, either using liposomes (Martinonet al., Eur J Immunol 23, 1719-1722 (1993)) or an intra-cytosolic invivo administration of the nucleic acid with a ballistic device (genegun) (Vassilev et al., Vaccine 19, 2012-2019 (2001)).

Since RNA molecules as such provide advantageous properties over DNA asdiscussed above, it is the object of the present invention to provide asuitable and efficient carrier for the transport of RNA into cells.Accordingly, the present invention provides a solution which allows RNAto transfect cells in an efficient manner.

This object of the present invention is achieved by the embodiments ofthe present invention as characterized by the claims. Particularly, theabove object is solved by a complexed RNA (molecule), comprising atleast one RNA (molecule), preferably an mRNA, complexed with one or moreoligopeptides, wherein the at least one oligopeptide has a length of 8to 15 amino acids, and wherein the at least one oligopeptide contains lArg residues, m Lys residues, n His residues, o Orn residues and x Xaaresidues positioned in any order within the at least one oligopeptidehaving the following empirical formula:

(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)  (formula I)

-   -   wherein        -   l+m+n+o+x=8-15, and        -   l, m, n or o independently of each other may be any number            selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,            14 or 15, provided that the overall content of Arg, Lys, His            and Orn represents at least 50%, e.g. at least 60% or 70%,            of all amino acids of the oligopeptide; and        -   Xaa may be any amino acid selected from native (=naturally            occurring) or non-native amino acids except of Arg, Lys, His            or Orn; and        -   x may be any number selected from 0, 1, 2, 3, 4, 5, 6, 7 or            8, provided, that the overall content of Xaa does not exceed            50%, e.g. not more than 40% or 30%, of all amino acids of            the oligopeptide.

In the context of the present invention, a complexed RNA is to beunderstood as an RNA (molecule) as defined herein, preferably an mRNA,which is complexed to the one or more oligopeptides according toempirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) byforming a non-covalent complex between RNA and oligopeptide(s). Herein,“non-covalent” means that a reversible association of RNA andoligopeptide is formed by non-covalent interactions of these molecules,wherein the molecules are associated together by any type of interactionof electrons, other than a covalent bond, e.g. by van der Waals-bonds,i.e. a weak electrostatic attraction arising from a nonspecificattractive force of the complexed molecules. Association of an RNA andat least one oligopeptide is in equilibrium with dissociation of thatcomplex. Intracellularly, without being bound to theory, the equilibriumappears to be shifted towards dissociated RNA and oligopeptide(s).

The at least one oligopeptide of the complexed RNA according to thepresent invention has a length of 8 to 15 amino acids, preferably alength of 8 to 14, 8 to 13, 8 to 12, or 9 to 12 or 9 to 11 amino acids,and more preferably a length of 8 to 10, 9 to 11, 10 to 12, 11 to 13, 12to 14 or 13 to 15 amino acids, or even more preferably may be selectedfrom a peptide of the above formula having a length of 8, 9, 10, 11, 12,13, 14 or 15 amino acids.

The oligopeptide of the complexed RNA according to the present inventionhas the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), as defined abovewherein l+m+n+o+x=8-15, and l, m, n or o independently of each other maybe any number selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14 or 15, or any range formed by two of these values, provided thatthe overall content of (the basic amino acids) Arg, Lys, His and/or Ornrepresents at least 50% (e.g. at least 51, 52, 53, 54, 55, 56, 57, 58,or 59%) at least 60% (e.g. at least 61, 62, 63, 64, 65, 66, 67, 68, or69%), at least 70% (e.g. at least 71, 72, 73, 74, 75, 76, 77, 78, or79%), at least 80% (e.g. at least 81, 82, 83, 84, 85, 86, 87, 88, or89%) at least 90% (e.g. at least 91, 92, 93, 94, 95, 96, 97, 98, or99%), or even 100% of all amino acids of the oligopeptide of thecomplexed RNA according to the present invention. The amino acids Arg,Lys, His and Orn (three letter code) are to be understood as the aminoacids arginine, lysine, histidine and omithine, respectively. In thiscontext, ornithine is an amino acid whose structure isNH₂—CH₂—CH₂—CH₂—CHNH₂—COOH. Ornithine was artificially incorporated asthe 21^(st) amino acid and does not belong to the “natively occurring”20 amino acids in the sense that ornithine is not an amino acid codedfor by DNA, and, accordingly, is not involved in primary proteinsynthesis. However, omithine is provided by enzymatic reaction startingfrom L-arginine. It is believed not to be a part of the genetic codebecause polypeptides containing unprotected ornithines undergospontaneous lactamization. Ornithine is to be regarded as a basic aminoacid since it is one of the products of the reaction of the enzymeArginase on L-arginine, creating urea.

According to a further preferred embodiment the (single) amino acids ofthe oligopeptide of the complexed RNA of the present invention, havingthe empirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)(formula I) as shown above, may occur in any frequency as defined abovefor the empirical formula, i.e. each basic amino acid (as well as Xaa)may occur in the above defined empirical formula within the abovedefined values or ranges, wherein any range may be formed from by two ofthe values as defined above. However, it is particularly preferred, ifthe content of the basic amino acid Arg in the above empricial formulais at least 10%, more preferably at least 20%, even more preferably atleast 30%, 40% or even 50%, even more preferably at least 60%, 70%, 80%90% or even 100% with respect to the entire empirical formula. Accordingto another particularly preferred embodiment the content of the basicamino acid Lys in the above empricial formula is at least 10%, morepreferably at least 20%, even more preferably at least 30%, 40% or even50%, even more preferably at least 60%, 70%, 80% 90% or even 100% withrespect to the entire empirical formula. According to a furtherparticularly preferred embodiment the content of the basic amino acidHis in the above empricial formula is at least 10%, more preferably atleast 20%, even more preferably at least 30%, 40% or even 50%, even morepreferably at least 60%, 70%, 80% 90% or even 100% with respect to theentire empirical formula. According to one other particularly preferredembodiment the content of the basic amino acid Orn in the aboveempricial formula is at least 10%, more preferably at least 20%, evenmore preferably at least 30%, 40% or even 50%, even more preferably atleast 60%, 70%, 80% 90% or even 100% with respect to the entireempirical formula. Any of the above defined contents, values or rangesof basic amino acids Arg, Lys, His and/or Orn as defined above may alsobe combined with each other, preferably leading to an overall content ofall basic amino acids of the oligopeptide of the complexed RNA of thepresent invention of at least 50% (at least 51, 52, 53, 54, 55, 56, 57,58, or 59%) at least 60% (at least 61, 62, 63, 64, 65, 66, 67, 68, or69%), at least 70% (at least 71, 72, 73, 74, 75, 76, 77, 78, or 79%), atleast 80% (at least 81, 82, 83, 84, 85, 86, 87, 88, or 89%) at least 90%(at least 91, 92, 93, 94, 95, 96, 97, 98, or 99%), or even 100%, asdefined initially.

The amino acids in the above formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), i.e. Arg, Lys, Hisand/or Orn may be furthermore be selected from the native (=naturallyoccurring) amino acids Arg, Lys, His and Orn or from non-native (=notnaturally occurring) amino acids derived from these amino acids. As anon-native (=not naturally occurring) amino acid derived from the aminoacids Arg, Lys, His and Orn, any known derivative of these amino acidsmay be used, which has been chemically modified, provided thesederivatives are not toxic for cells or organisms, when provided with theabove oligopeptide. (Such derivatives of amino acids are distributed bydifferent companies; see e.g. Sigma Aldrich (seehttp://www.sigmaaldrich.com).

Furthermore, the oligopeptide of the complexed RNA according to thepresent invention may contain an amino acid Xaa in the above empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), which may beany amino acid selected from native (=naturally occurring) or non-native(=not naturally occurring) amino acids except of Arg, Lys, His or Orn.Preferably, Xaa may be selected, without being limited thereto, fromnaturally occurring neutral (and hydrophobic) amino acids, i.e. aminoacids, which have neutral (and hydrophobic) side chains, such as alanine(Ala), valine (Val), leucine (Leu), isoleucine (Ile), proline (Pro),tryptophane (Trp), phenylalanine (Phe), or methionine (Met), and/or fromnaturally occurring neutral (and polar) amino acids, i.e. amino acids,which have neutral (and polar) side chains, such as glycine (Gly),serine (ser), threonine (Thr), tyrosine (Tyr), cysteine (Cys),asparagine (Asn), or glutamine (Glu), and/or from naturally occurringacidic amino acids, i.e. amino acids, which have acidic side chains,such as aspartic acid (Asp) or glutamic acid (Glu). Preferably theoligopeptide of the complexed RNA according to the present invention maycontain an amino acid Xaa in the above empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), which is selectedfrom amino acids having no acidic side chain. Even more preferably, Xaain empirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)is selected from amino acids having a neutral side chain, i.e. fromamino acids, which have a neutral (and hydrophobic) side chain and/orfrom amino acids, which have a neutral (and polar) side chain, asdefined above. Additionally, any known derivative of amino acids may beused for Xaa in the above empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), i.e. amino acids,which have been chemically modified, provided these derivatives are nottoxic for cells or organisms, when provided with the above oligopeptide.(Such derivatives of amino acids are distributed by different companies,see e.g. Sigma Aldrich (see http://www.sigmaaldrich.com). Xaa istypically present in the above formula in a content of 0-30%, 0-40% or0-50% of all amino acids of the entire oligopeptide sequence, i.e. theoverall content of Xaa may not exceed 30%, 40% or 50% of all amino acidsof the entire oligopeptide sequence, preferably it may not exceed 20%,even more preferably not 10%, and most preferably not 5% of all aminoacids of the entire oligopeptide sequence. Thus, x in the empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown abovemay be any number selected from 0, 1, 2, 3, 4, 5, 6, 7 or 8, provided,that the content of Xaa does not exceed the above indicated value of 30%(or less), 40% or 50% of all entire amino acids of the oligopeptide ofthe complexed RNA.

Typically, the amino acids Arg, Lys, His, Orn and Xaa of theoligopeptide of the complexed RNA according to the present invention,having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as indicated above,may be positioned at any position of the oligopeptide sequence.Accordingly, empirical formula (I) does not determine any specific orderof amino acids, but is rather intended to reflect the type of aminoacids and their frequency of occurrence in the peptide, indicating thatthe peptide chain contains a number of l Arg residues, m Lys residues, nHis residues, o Orn residues and x Xaa residues, without specifying anyorder of these residues within the peptide chain.

However, it is preferred, that the above oligopeptide comprises aminoacids at one or, preferably, both terminal ends, which do not comprisean acidic side chain. More preferably, the above oligopeptide sequencecomprises neutral or basic amino acids at one or, preferably, bothterminal ends, even more preferably basic amino acids at one or bothterminal ends. In a further preferred embodiment, the oligopeptideaccording to the general formula given above contains at least two, morepreferably at least three, at least four or even at least five terminalbasic residues, in particular Arg, Orn or Lys, at either terminus.According to just another preferred embodiment, the oligopeptideaccording to the general formula given above preferably comprises nocationic amino acids (i.e. no Arg, Orn or Lys) at one or, preferably, atboth terminal ends, even more preferably no cationic amino acids (i.e.no Arg, Orn or Lys) at both terminal ends. In other words, one, or morepreferably both, terminal ends of the oligopeptide according to thegeneral formula given above may comprise any non-cationic amino acid asdefined herein, provided that such non-cationic amino acid is selectedfrom an amino acid except Arg, Orn or Lys or any variant or derivativeof these cationic amino acids. The terminal ends may comprise e.g. one,at least two, at least three, at least four, at least five or even morebasic non-cationic residues as defined above starting from the N- and/orC-terminal end of the particular sequence.

According to a further preferred embodiment, one or both terminal endsof the oligopeptide of the complexed RNA according to the presentinvention may comprise at least one histidine residues at one or both ofits terminal ends, e.g. the oligopeptide of the complexed RNA accordingto the present invention may comprise one, two, three or more histidineresidues in consecutive order at one or both terminal ends, providedthat the overall length of the oligopeptide is limited to 8 to 15 aminoacids as defined above.

Additionally, Xaa residues of the oligopeptide of the complexed RNAaccording to the present invention are typically separated from eachother by at least one Arg, Lys, His or Orn. Such a separation of Xaaresidues preferably avoids clusters of non-basic amino acids in theoligopeptide, as such non-basic clusters may reduce the advantageousproperties of the oligopeptide as a carrier peptide for the complexedRNA according to the present invention.

However, basic amino acid residues of the oligopeptide of the complexedRNA according to the formula given above are selected from Arg, Lys. Hisor Orn as defined above and typically occur in a cluster of at least 2,preferably at least 3, 4, 5, or even 6 or more basic amino acids asdefined herein. According to a particularly preferred embodiment, suchclusters may also comprise 6, 7, 8, 9, 10, 11, 12, 13, 14 or even 15amino acids. Such a cluster of basic amino acids, preferably a clusterof at least 3, 4, 5, or even 6 or more basic amino acids preferablycreates a basic surface or binding region within the oligopeptide, whichprovides advantageous properties to the oligopeptide as a carrierpeptide for the complexed RNA according to the present invention.

According to a further preferred embodiment the oligopeptide of thecomplexed RNA of the present invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula I) as shownabove, may be, without being restricted thereto, selected from thefollowing subgroup of formulae:

Arg₈, Arg₉, Arg₁₀, Arg₁₁, Arg₁₂, Arg₁₃, Arg₁₄, Arg₁₅, (SEQ ID NOs: 1-8);Lys₈, Lys₉, Lys₁₀, Lys₁₁, Lys₁₂, Lys₁₃, Lys₁₄, Lys₁₅, (SEQ ID NOs:9-16);His₈, His₉, His₁₀, His₁₁, His₁₂, His₁₃, His₁₄, His₁₅, (SEQ ID NOs:17-24);Orn₈, Orn₉, Orn₁₀, Orn₁₁, Orn₁₂, Orn₁₃, Orn₁₄, Orn₁₅, (SEQ ID NOs:25-32);

According to a further preferred embodiment the oligopeptide of thecomplexed RNA of the present invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula I) as shownabove, may be, without being restricted thereto, selected from followingsubgroup. This subgroup exemplarily defines specific inventiveoligopeptides, which fall under empirical formula I as defined above,wherein the following formulae (as with empirical formula (I)) do notspecify any amino acid order, but are intended to reflect empiricalformulae by exclusively specifying the (number of) amino acids ascomponents of the respective peptide. Accordingly, empirical formulaArg₍₇₋₁₄₎Lys₁ is intended to mean that peptides falling under thisformula contain 7 to 14 Arg residues and 1 Lys residue of whatsoeverorder. If the peptides contain 7 Arg residues and 1 Lys residue, allvariants having 7 Arg residues and 1 Lys residue are encompassed. TheLys residue may therefore be positioned anywhere in the e.g. 8 aminoacid long sequence composed of 7 Arg and 1 Lys residues. The subgrouppreferably comprises:

Arg₍₇₋₁₄₎Lys₁, Arg₍₇₋₁₄₎His₁, Arg₍₇₋₁₄₎Orn₁, Lys₍₇₋₁₄₎His₁,Lys₍₇₋₁₄₎Orn₁, His₍₇₋₁₄₎Orn₁;Arg₍₆₋₁₃₎Lys₂, Arg₍₆₋₁₃₎His₂, Arg₍₆₋₁₃₎Orn₂, Lys₍₆₋₁₃₎His₂,Lys₍₆₋₁₃₎Orn₂, His₍₆₋₁₃₎Orn₂;Arg₍₅₋₁₂₎Lys₃, Arg₍₅₋₁₂₎His₃, Arg₍₅₋₁₂₎Orn₃, Lys₍₅₋₁₂₎His₃,Lys₍₅₋₁₂₎Orn₃, His₍₅₋₁₂₎Orn₃;Arg₍₄₋₁₁₎Lys₄, Arg₍₄₋₁₁₎His₄, Arg₍₄₋₁₁₎Orn₄, Lys₍₄₋₁₁₎His₄,Lys₍₄₋₁₁₎Orn₄, His₍₄₋₁₁₎Orn₄;Arg₍₃₋₁₀₎Lys₅, Arg₍₃₋₁₀₎His₅, Arg₍₃₋₁₀₎Orn₅, Lys₍₃₋₁₀₎His₅,Lys₍₃₋₁₀₎Orn₅, His₍₃₋₁₀₎Orn₅;Arg₍₂₋₉₎Lys₆, Arg₍₂₋₉₎His₆, Arg₍₂₋₉₎Orn₆, Lys₍₂₋₉₎His₆, Lys₍₂₋₉₎Orn₆,His₍₂₋₉₎Orn₆;Arg₍₁₋₈₎Lys₇, Arg₍₁₋₈₎His₇, Arg₍₁₋₈₎Orn₇, Lys₍₁₋₈₎His₇, Lys₍₁₋₈₎Orn₇,His₍₁₋₈₎Orn₇;Arg₍₆₋₁₃₎Lys₁His₁, Arg₍₆₋₁₃₎Lys₁Orn₁, Arg₍₆₋₁₃₎His₁Orn₁,Arg₁Lys₍₆₋₁₃₎His₁, Arg₁Lys₍₆₋₁₃₎Orn₁, Lys₍₆₋₁₃₎His₁Orn₁,Arg₁Lys₁His₍₆₋₁₃₎, Arg₁His₍₆₋₁₃₎Orn₁, Lys₁His₍₆₋₁₃₎Orn₁;Arg₍₅₋₁₂₎Lys₂His₁, Arg₍₅₋₁₂₎Lys₁His₂, Arg₍₅₋₁₂₎Lys₂Orn₁,Arg₍₅₋₁₂₎Lys₁Orn₂, Arg₍₅₋₁₂₎His₂Orn₁, Arg₍₅₋₁₂₎His₁Orn₂,Arg₂Lys₍₅₋₁₂₎His₁, Arg₁Lys₍₅₋₁₂₎His₂, Arg₂Lys₍₅₋₁₂₎Orn₁,Arg₁Lys₍₅₋₁₂₎Orn₂, Lys₍₅₋₁₂₎His₂Orn₁, Lys₍₅₋₁₂₎His₁Orn₂,Arg₂Lys₁His₍₅₋₁₂₎, Arg₁Lys₂His₍₅₋₁₂₎, Arg₂His₍₅₋₁₂₎Orn₁,Arg₁His₍₅₋₁₂₎Orn₂, Lys₂His₍₅₋₁₂₎Orn₁, Lys₁His₍₅₋₁₂₎Orn₂;Arg₍₄₋₁₁₎Lys₃His₁, Arg₍₄₋₁₁₎Lys₂His₂, Arg₍₄₋₁₁₎Lys₁His₃,Arg₍₄₋₁₁₎Lys₃Orn₁, Arg₍₄₋₁₁₎Lys₂Orn₂, Arg₍₄₋₁₁₎Lys₁Orn₃,Arg₍₄₋₁₁₎His₃Orn₁, Arg₍₄₋₁₁₎His₂Orn₂, Arg₍₄₋₁₁₎His₁Orn₃,Arg₃Lys₍₄₋₁₁₎His₁, Arg₂Lys₍₄₋₁₁₎His₂, Arg₁Lys₍₄₋₁₁₎His₃,Arg₃Lys₍₄₋₁₁₎Orn₁, Arg₂Lys₍₄₋₁₁₎Orn₂, Arg₁Lys₍₄₋₁₁₎Orn₃,Lys₍₄₋₁₁₎His₃Orn₁, Lys₍₄₋₁₁₎His₂Orn₂, Lys₍₄₋₁₁₎His₁Orn₃,Arg₃Lys₁His₍₄₋₁₁₎, Arg₂Lys₂His₍₄₋₁₁₎, Arg₁Lys₃His₍₄₋₁₁₎,Arg₃His₍₄₋₁₁₎Orn₁, Arg₂His₍₄₋₁₁₎Orn₂, Arg₁His₍₄₋₁₁₎Orn₃,Lys₃His₍₄₋₁₁₎Orn₁, Lys₂His₍₄₋₁₁₎Orn₂, Lys₁His₍₄₋₁₁₎Orn₃;Arg₍₃₋₁₀₎Lys₄His₁, Arg₍₃₋₁₀₎Lys₃His₂, Arg₍₃₋₁₀₎Lys₂His₃,Arg₍₃₋₁₀₎Lys₁His₄, Arg₍₃₋₁₀₎Lys₄Orn₁, Arg₍₃₋₁₀₎Lys₃Orn₂,Arg₍₃₋₁₀₎Lys₂Orn₃, Arg₍₃₋₁₀₎Lys₁Orn₄, Arg₍₃₋₁₀₎His₄Orn₁,Arg₍₃₋₁₀₎His₃Orn₂, Arg₍₃₋₁₀₎His₂Orn₃, Arg₍₃₋₁₀₎His₁Orn₄,Arg₄Lys₍₃₋₁₀₎His₁, Arg₃Lys₍₃₋₁₀₎His₂, Arg₂Lys₍₃₋₁₀₎His₃,Arg₁Lys₍₃₋₁₀₎His₄, Arg₄Lys₍₃₋₁₀₎Orn₁, Arg₃Lys₍₃₋₁₀₎Orn₂,Arg₂Lys₍₃₋₁₀₎Orn₃, Arg₁Lys₍₃₋₁₀₎Orn₄, Lys₍₃₋₁₀₎His₄Orn₁,Lys₍₃₋₁₀₎His₃Orn₂, Lys₍₃₋₁₀₎His₂Orn₃, Lys₍₃₋₁₀₎His₁Orn₄,Arg₄Lys₁His₍₃₋₁₀₎, Arg₃Lys₂His₍₃₋₁₀₎, Arg₂Lys₃His₍₃₋₁₀₎,Arg₁Lys₄His₍₃₋₁₀₎, Arg₄His₍₃₋₁₀₎Orn₁, Arg₃His₍₃₋₁₀₎Orn₂,Arg₂His₍₃₋₁₀₎Orn₃, Arg₁His₍₃₋₁₀₎Orn₄, Lys₄His₍₃₋₁₀₎Orn₁,Lys₃His₍₃₋₁₀₎Orn₂, Lys₂His₍₃₋₁₀₎Orn₃, Lys₁His₍₃₋₁₀₎Orn₄;Arg₍₂₋₉₎Lys₅His₁, Arg₍₂₋₉₎Lys₄His₂, Arg₍₂₋₉₎Lys₃His₃, Arg₍₂₋₉₎Lys₂His₄,Arg₍₂₋₉₎Lys₁His₅, Arg₍₂₋₉₎Lys₅Orn₁, Arg₍₂₋₉₎Lys₄Orn₂, Arg₍₂₋₉₎Lys₃Orn₃,Arg₍₂₋₉₎Lys₂Orn₄, Arg₍₂₋₉₎Lys₁Orn₅, Arg₍₂₋₉₎His₅Orn₁, Arg₍₂₋₉₎His₄Orn₂,Arg₍₂₋₉₎His₃Orn₃, Arg₍₂₋₉₎His₂Orn₄, Arg₍₂₋₉₎His₁Orn₅, Arg₅Lys₍₂₋₉₎His₁,Arg₄Lys₍₂₋₉₎His₂, Arg₃Lys₍₂₋₉₎His₃, Arg₂Lys₍₂₋₉₎His₄, Arg₁Lys₍₂₋₉₎His₅,Arg₅Lys₍₂₋₉₎Orn₁, Arg₄Lys₍₂₋₉₎Orn₂, Arg₃Lys₍₂₋₉₎Orn₃, Arg₂Lys₍₂₋₉₎Orn₄,Arg₁Lys₍₂₋₉₎Orn₅, Lys₍₂₋₉₎His₅Orn₁, Lys₍₂₋₉₎His₄Orn₂, Lys₍₂₋₉₎His₃Orn₃,Lys₍₂₋₉₎His₂Orn₄, Lys₍₂₋₉₎His₁Orn₅, Arg₅Lys₁His₍₂₋₉₎, Arg₄Lys₂His₍₂₋₉₎,Arg₃Lys₃His₍₂₋₉₎, Arg₂Lys₄His₍₂₋₉₎, Arg₁Lys₅His₍₂₋₉₎, Arg₅His₍₂₋₉₎Orn₁,Arg₄His₍₂₋₉₎Orn₂, Arg₃His₍₂₋₉₎Orn₃, Arg₂His₍₂₋₉₎Orn₄, Arg₁His₍₂₋₉₎Orn₅,Lys₅His₍₂₋₉₎Orn₁, Lys₄His₍₂₋₉₎Orn₂, Lys₃His₍₂₋₉₎Orn₃, Lys₂His₍₂₋₉₎Orn₄,Lys₁His₍₂₋₉₎Orn₅;Arg₍₁₋₈₎Lys₆His₁, Arg₍₁₋₈₎Lys₅His₂, Arg₍₁₋₈₎Lys₄His₃, Arg₍₁₋₈₎Lys₃His₄,Arg₍₁₋₈₎Lys₂His₅, Arg₍₁₋₈₎Lys₁His₆, Arg₍₁₋₈₎Lys₆Orn₁, Arg₍₁₋₈₎Lys₅Orn₂,Arg₍₁₋₈₎Lys₄Orn₃, Arg₍₁₋₈₎Lys₃Orn₄, Arg₍₁₋₈₎Lys₂Orn₅, Arg₍₁₋₈₎Lys₁Orn₆,Arg₍₁₋₈₎His₆Orn₁, Arg₍₁₋₈₎His₅Orn₂, Arg₍₁₋₈₎His₄Orn₃, Arg₍₁₋₈₎His₃Orn₄,Arg₍₁₋₈₎His₂Orn₅, Arg₍₁₋₈₎His₁Orn₆, Arg₆Lys₍₁₋₈₎His₁, Arg₅Lys₍₁₋₈₎His₂,Arg₄Lys₍₁₋₈₎His₃, Arg₃Lys₍₁₋₈₎His₄, Arg₂Lys₍₁₋₈₎His₅, Arg₁Lys₍₁₋₈₎His₆,Arg₆Lys₍₁₋₈₎Orn₁, Arg₅Lys₍₁₋₈₎Orn₂, Arg₄Lys₍₁₋₈₎Orn₃, Arg₃Lys₍₁₋₈₎Orn₄,Arg₂Lys₍₁₋₈₎Orn₅, Arg₁Lys₍₁₋₈₎Orn₆, Lys₍₁₋₈₎His₆Orn₁, Lys₍₁₋₈₎His₅Orn₂,Lys₍₁₋₈₎His₄Orn₃, Lys₍₁₋₈₎His₃Orn₄, Lys₍₁₋₈₎His₂Orn₅, Lys₍₁₋₈₎His₁Orn₆,Arg₆Lys₁His₍₁₋₈₎, Arg₅Lys₂His₍₁₋₈₎, Arg₄Lys₃His₍₁₋₈₎, Arg₃Lys₄His₍₁₋₈₎,Arg₂Lys₅His₍₁₋₈₎, Arg₁Lys₆His₍₁₋₈₎, Arg₆His₍₁₋₈₎Orn₁, Arg₅His₍₁₋₈₎Orn₂,Arg₄His₍₁₋₈₎Orn₃, Arg₃His₍₁₋₈₎Orn₄, Arg₂His₍₁₋₈₎Orn₅, Arg₁His₍₁₋₈₎Orn₆,Lys₆His₍₁₋₈₎Orn₁, Lys₅His₍₁₋₈₎Orn₂, Lys₄His₍₁₋₈₎Orn₃, Lys₃His₍₁₋₈₎Orn₄,Lys₂His₍₁₋₈₎Orn₅, Lys₁His₍₁₋₈₎Orn₆;Arg₍₅₋₁₂₎Lys₁His₁Orn₁, Arg₁Lys₍₅₋₁₂₎His₁Orn₁, Arg₁Lys₁His₍₅₋₁₂₎Orn₁,Arg₁Lys₁His₁Orn₍₅₋₁₂₎;Arg₍₄₋₁₁₎Lys₂His₁Orn₁, Arg₍₄₋₁₁₎Lys₁His₂Orn₁, Arg₍₄₋₁₁₎Lys₁His₁Orn₂,Arg₂Lys₍₄₋₁₁₎His₁Orn₁, Arg₁Lys₍₄₋₁₁₎His₂Orn₁, Arg₁Lys₍₄₋₁₁₎His₁Orn₂,Arg₂Lys₁His₍₄₋₁₁₎Orn₁, Arg₁Lys₂His₍₄₋₁₁₎Orn₁, Arg₁Lys₁His₍₄₋₁₁₎Orn₂,Arg₂Lys₁His₁Orn₍₄₋₁₁₎, Arg₁Lys₂His₁Orn₍₄₋₁₁₎, Arg₁Lys₁His₂Orn₍₄₋₁₁₎;Arg₍₃₋₁₀₎Lys₃His₁Orn₁, Arg₍₃₋₁₀₎Lys₂His₂Orn₁, Arg₍₃₋₁₀₎Lys₂His₁Orn₂,Arg₍₃₋₁₀₎Lys₁His₂Orn₂, Arg₍₃₋ ₁₀₎Lys₁His₁Orn₃, Arg₃Lys₍₃₋₁₀₎His₁Orn₁,Arg₂Lys₍₃₋₁₀₎His₂Orn₁, Arg₂Lys₍₃₋₁₀₎His₁Orn₂, Arg₁Lys₍₃₋₁₀₎His₂Orn₂,Arg₁Lys₍₃₋₁₀₎His₁Orn₃, Arg₃Lys₁His₍₃₋₁₀₎Orn₁, Arg₂Lys₂His₍₃₋₁₀₎Orn₁,Arg₂Lys₁His₍₃₋₁₀₎Orn₂, Arg₁Lys₂His₍₃₋₁₀₎Orn₂, Arg₁Lys₁His₍₃₋₁₀₎Orn₃,Arg₃Lys₁His₁Orn₍₃₋₁₀₎, Arg₂Lys₂His₁Orn₍₃₋₁₀₎, Arg₂Lys₁His₂Orn₍₃₋₁₀₎,Arg₁Lys₂His₂Orn₍₃₋₁₀₎, Arg₁Lys₁His₃Orn₍₃₋₁₀₎;Arg₍₂₋₉₎Lys₄His₁Orn₁, Arg₍₂₋₉₎Lys₁His₄Orn₁, Arg₍₂₋₉₎Lys₁His₁Orn₄,Arg₍₂₋₉₎Lys₃His₂Orn₁, Arg₍₂₋₉₎Lys₃His₁Orn₂, Arg₍₂₋₉₎Lys₂His₃Orn₁,Arg₍₂₋₉₎Lys₂His₁Orn₃, Arg₍₂₋₉₎Lys₁His₂Orn₃, Arg₍₂₋₉₎Lys₁His₃Orn₂,Arg₍₂₋₉₎Lys₂His₂Orn₂, Arg₄Lys₍₂₋₉₎His₁Orn₁, Arg₁Lys₍₂₋₉₎His₄Orn₁,Arg₁Lys₍₂₋₉₎His₁Orn₄, Arg₃Lys₍₂₋₉₎His₂Orn₁, Arg₃Lys₍₂₋₉₎His₁Orn₂,Arg₂Lys₍₂₋₉₎His₃Orn₁, Arg₂Lys₍₂₋₉₎His₁Orn₃, Arg₁Lys₍₂₋₉₎His₂Orn₃,Arg₁Lys₍₂₋₉₎His₃Orn₂, Arg₂Lys₍₂₋₉₎His₂Orn₂, Arg₄Lys₁His₍₂₋₉₎Orn₁,Arg₁Lys₄His₍₂₋₉₎Orn₁, Arg₁Lys₁His₍₂₋₉₎Orn₄, Arg₃Lys₂His₍₂₋₉₎Orn₁,Arg₃Lys₁His₍₂₋₉₎Orn₂, Arg₂Lys₃His₍₂₋₉₎Orn₁, Arg₂Lys₁His₍₂₋₉₎Orn₃,Arg₁Lys₂His₍₂₋₉₎Orn₃, Arg₁Lys₃His₍₂₋₉₎Orn₂, Arg₂Lys₂His₍₂₋₉₎Orn₂,Arg₄Lys₁His₁Orn₍₂₋₉₎, Arg₁Lys₄His₁Orn₍₂₋₉₎, Arg₁Lys₁His₄Orn₍₂₋₉₎,Arg₃Lys₂His₁Orn₍₂₋₉₎, Arg₃Lys₁His₂Orn₍₂₋₉₎, Arg₂Lys₃His₁Orn₍₂₋₉₎,Arg₂Lys₁His₃Orn₍₂₋₉₎, Arg₁Lys₂His₃Orn₍₂₋₉₎, Arg₁Lys₃His₂Orn₍₂₋₉₎,Arg₂Lys₂His₂Orn₍₂₋₉₎;Arg₍₁₋₈₎Lys₅His₁Orn₁, Arg₍₁₋₈₎Lys₁His₅Orn₁, Arg₍₁₋₈₎Lys₁His₁Orn₅,Arg₍₁₋₈₎Lys₄His₂Orn₁, Arg₍₁₋₈₎Lys₂His₄Orn₁, Arg₍₁₋₈₎Lys₂His₁Orn₄,Arg₍₁₋₈₎Lys₁His₂Orn₄, Arg₍₁₋₈₎Lys₁His₄Orn₂, Arg₍₁₋₈₎Lys₄His₁Orn₂,Arg₍₁₋₈₎Lys₃His₃Orn₁, Arg₍₁₋₈₎Lys₃His₁Orn₃, Arg₍₁₋₈₎Lys₁His₃Orn₃,Arg₅Lys₍₁₋₈₎His₁Orn₁, Arg₁Lys₍₁₋₈₎His₅Orn₁, Arg₁Lys₍₁₋₈₎His₁Orn₅,Arg₄Lys₍₁₋₈₎His₂Orn₁, Arg₂Lys₍₁₋₈₎His₄Orn₁, Arg₂Lys₍₁₋₈₎His₁Orn₄,Arg₁Lys₍₁₋₈₎His₂Orn₄, Arg₁Lys₍₁₋₈₎His₄Orn₂, Arg₄Lys₍₁₋₈₎His₁Orn₂,Arg₃Lys₍₁₋₈₎His₃Orn₁, Arg₃Lys₍₁₋₈₎His₁Orn₃, Arg₁Lys₍₁₋₈₎His₃Orn₃,Arg₅Lys₁His₍₁₋₈₎Orn₁, Arg₁Lys₅His₍₁₋₈₎Orn₁, Arg₁Lys₁His₍₁₋₈₎Orn₅,Arg₄Lys₂His₍₁₋₈₎Orn₁, Arg₂Lys₄His₍₁₋₈₎Orn₁, Arg₂Lys₁His₍₁₋₈₎Orn₄,Arg₁Lys₂His₍₁₋₈₎Orn₄, Arg₁Lys₄His₍₁₋₈₎Orn₂, Arg₄Lys₁His₍₁₋₈₎Orn₂,Arg₃Lys₃His₍₁₋₈₎Orn₁, Arg₃Lys₁His₍₁₋₈₎Orn₃, Arg₁Lys₃His₍₁₋₈₎Orn₃,Arg₅Lys₁His₁Orn₍₁₋₈₎, Arg₁Lys₅His₁Orn₍₁₋₈₎, Arg₁Lys₁His₅Orn₍₁₋₈₎,Arg₄Lys₂His₁Orn₍₁₋₈₎, Arg₂Lys₄His₁Orn₍₁₋₈₎, Arg₂Lys₁His₄Orn₍₁₋₈₎,Arg₁Lys₂His₄Orn₍₁₋₈₎, Arg₁Lys₄His₂Orn₍₁₋₈₎, Arg₄Lys₁His₂Orn₍₁₋₈₎,Arg₃Lys₃His₁Orn₍₁₋₈₎, Arg₃Lys₁His₃Orn₍₁₋₈₎, Arg₁Lys₃His₃Orn₍₁₋₈₎;

According to one preferred embodiment, the oligopeptide of the complexedRNA of the present invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, isselected from the subgroup consisting of: Arg₈, Arg₉, Arg₁₀, Arg₁₁,Arg₁₂, Arg₁₃, Arg₁₄, Arg₁₅, (SEQ ID NOs: 1-8); Lys₈, Lys₉, Lys₁₀, Lys₁₁,Lys₁₂, Lys₁₃, Lys₁₄, Lys₁₅, (SEQ ID NOs: 9-16); His₈, His₉, His₁₀,His₁₁, His₁₂, His₁₃, His₁₄, His₁₅, (SEQ ID NOs: 17-24); or Orn₈, Orn₉,Orn₁₀, Orn₁₁, Orn₁₂, Orn₁₃, Orn₁₄, Orn₁₅, (SEQ ID NOs: 25-32).

According to another preferred embodiment, the oligopeptide of thecomplexed RNA of the present invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, isselected from the subgroup consisting of general formulas Arg, (alsotermed R9), Arg₉His₃ (also termed R9H3), His₃Arg₉His₃ (also termedH3R9H3), TyrSerSerArg₉SerSerTyr (also termed YSSR9SSY),His₃Arg₉SerSerTyr (also termed H3R9SSY), (ArgLysHis)₄ (also termed(RKH)4), Tyr(ArgLysHis)₂Arg (also termed Y(RKH)2R). Even morepreferably, these general formulas are defined as follows:

Arg₉: (SEQ ID NO: 2) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg Arg₉His₃:(SEQ ID NO: 39) Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-HisHis₃Arg₉His₃: (SEQ ID NO: 40)His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- His-His-HisTyrSerSerArg₉SerSerTyr: (SEQ ID NO: 41)Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-TyrHis₃Arg₉SerSerTyr: (SEQ ID NO: 42)His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr(ArgLysHis)₄: (SEQ ID NO: 43)Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His Tyr(ArgLysHis)₂Arg:(SEQ ID NO: 44) Tyr-Arg-Lys-His-Arg-Lys-His-Arg 

The at least one oligopeptide of the complexed RNA (molecule) of thepresent invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, may beadditionally modified. Modifications in the context of the presentinvention typically comprise any modification suitable for peptides,provided that these modifications do not interfere with the transfectioncapabilities of the resulting complexed RNA.

Typical modifications may thus include e.g. the use of modified aminoacids as defined above. Furthermore, the terminal amino acid residues ofthe oligopeptide, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, withtheir carboxy (C-terminus) and their amino (N-terminus) groups (as wellas carboxy or amide amino acid side chain groups, see above) may bepresent in their protected (e.g. the C terminus protected by an amidegroup) and/or unprotected form, using appropriate amino or carboxylprotecting groups. Also, acid-addition salts of the oligopeptide, havingthe empirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x),as shown above, may be used. Common acid addition salts are hydrohalicacid salts, i.e., HBr, HI, or more preferably, HCl.

PEGylation of terminal or side chain carboxyl groups or theepsilon-amino group of lysine occurring in the oligopeptide, having theempirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) asshown above, confers resistance to agglomeration and serum degradationand is also within the scope of the present invention.

The at least one oligopeptide of the complexed RNA (molecule) of thepresent invention, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, mayfurthermore be modified to bind to or be coupled to at least onespecific ligand, wherein the at least one specific ligand may be boundto or coupled to one or both terminal ends of the at least oneoligopeptide. The at least one specific ligand bound to or coupled toone or both terminal ends of the oligopeptide may be identical ordifferent and may be selected from any compound capable to bind to orinteract with a receptor or a protein or a protein/receptor complex,e.g. at the cell surface, e, e.g., without being limited thereto,RGD-peptide, transferrin or mannose, etc.

Other preferred modifications resulting in derivatives of theoligopeptide, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, arebased on carbohydrates and/or lipids which may be covalently coupled tothe oligopeptide. It is preferred to couple carbohydrates and/or lipidsto serine, threonine, asparagine, glutamine or tyrosine or glutamate oraspartate via their reactive side chain moieties. Alternatively,carbohydrates and/or lipids may also be linked to the terminal moietiesof the oligopeptide as defined herein. Furthermore, the oligopeptide maybe coupled to a functionally different peptide or protein moiety, whichmay also stabilize the oligopeptide and/or may serve to improve thetransport properties of oligopeptide in body fluids, in particularblood. Suitable peptides or proteins may e.g. be selected from albumin,transferrin etc., which may be directly coupled to the oligopeptide,having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, or viaa peptide or organic linker sequence. Preferably, these peptides orproteins are linked to one of the termini of the oligopeptide.

In this context, it is to be noted that a modification of theoligopeptide with lipids, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, doestypically not include the use of (saturated or non-saturated) fattyacids, particularly not the use of long chain (saturated ornon-saturated) fatty acids (in particular with a chain length of >C₁₂,>C₁₄ or >C₁₆). Thus, in the context of the present invention,modification of the oligopeptide with fatty acids, having the empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shownabove, does not form an integral part of the present invention. However,if fatty acids are used at all to modify the carrier peptide, thex maybe selected, without being limited thereto, from the group comprisinge.g. butanoic fatty acid (butyric fatty acid), pentanoic fatty acid(valeric fatty acid), hexanoic fatty acid (caproic fatty acid), octanoicfatty acid (caprylic fatty acid), nonanoic fatty acid (pelargonic fattyacid), decanoic fatty acid (capric fatty acid), dodecanoic fatty acid(lauric fatty acid), tetradecanoic fatty acid (myristic fatty acid),hexadecanoic fatty acid (palmitic fatty acid), heptadecanoic fatty acid(margaric (daturic) fatty acid), octadecanoic fatty acid (stearic fattyacid), eicosanoic fatty acid (arachidic fatty acid), docosanoic fattyacid (behenic fatty acid), tetracosanoic fatty acid (lignoceric fattyacid), hexacosanoic fatty acid (cerotic fatty acid), heptacosanoic fattyacid (carboceric fatty acid), octacosanoic fatty acid (montanic fattyacid), triacontanoic fatty acid (melissic fatty acid), dotriacontanoicfatty acid (lacceroic fatty acid), tritriacontanoic fatty acid(ceromelissic (psyllic) fatty acid), tetratriacontanoic fatty acid(geddic fatty acid), pentatriacontanoic fatty acid (ceroplastic fattyacid), etc., or their non-saturated analogs. As a particular example,the present invention does typically not include the use of octadecanoicfatty acid (stearic fatty acid) or its non-saturated analogs formodification of the carrier peptides of formula I, i.e. typically nostearylated oligopeptides of formula I may be used herein forcomplexation of the RNA component of the inventive complex.

In order to circumvent the problem of degradation of the oligopeptide,having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above,according to another embodiment of the present invention a retro-inversoisomer of the above oligopeptide composed of D amino acids or at leastpartially composed of D amino acids may be used. The term “retro-inversoisomer” refers to an isomer of a linear peptide in which the directionof the sequence is reversed and the chirality of each amino acid residueis inverted (see, e.g., Jameson et al., Nature, 368, 744-746 (1994);Brady et al., Nature, 368, 692-693 (1994)). With respect to the parentpeptide, the retro-inverso peptide is assembled in reverse order ofamino acids, typically with F-moc amino acid derivatives. Typically, thecrude peptides may be purified by reversed phase HPLC.

Other modifications, which may be introduced into the oligopeptide,having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) as shown above, relateto modifications of the peptide backbone. Preferably, the modifiedoligopeptides are scaffold mimetics. Their backbone is different fromthe natural occurring backbone, while their side-chain structures areidentical with the oligopeptides or their fragments, variants orderivatives. In general, scaffold mimetics exhibit a modification of oneor more of the backbone chain members (NH, CH, CO), either assubstitution (preferably) or as an insertion. Substituents are e.g. (I)—O—, —S—, or —CH₂— instead of —NH—; (II) —N—, C-Alkyl-, or —BH— insteadof —CHR— and (III) —CS—, —CH₂—, —SO_(n)—, —P═O(OH)—, or —B(OH)— insteadof —CO—. A peptide mimetic of an oligopeptide, having the empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), as definedherein, may be a combination of each of these modifications. Inparticular, modifications of each the groups I, II and III may becombined. In a peptide mimetic each backbone chain member may bemodified or, alternatively, only a certain number of chain members maybe exchanged for a non-naturally occurring moiety. Preferably, allbackbone chain members of an oligopeptide, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), as defined herein, ofeither —NH—, —CHR— or CO are exchanged for another non-naturallyoccurring group. In case the amide bond (—NH—CO—) of the oligopeptidebackbone is substituted (in the entire molecule or at least in onesingle position), preferable substitution moieties are bioisosteric,e.g. retro-inverse amide bonds (—CO—NH—), hydroxyl ethylene(—CH(OH)—CH₂—), alkene (CH₂═CH—), carba (CH₂—CH₂—) and/or—P—O(OH)—CH₂—). Alternatively, backbone chain elongation by insertionsmay occur in a scaffold mimetic of the oligopeptide, having theempirical formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) asdefined herein, e.g. by moieties flanking the C-alpha atom. On eitherside of the C-alpha atom e.g. —O—, —S—, —CH—, —NH— may be inserted.

Particularly preferred are oligocarbamate peptide backbone structure ofthe oligopeptide, having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), as defined herein.Thereby amide bond may be replaced by a carbamate moiety. The monomericN-protected amino alkyl carbonates are accessible via the correspondingamino acids or amino alcohols. They are converted into active esters,e.g. p-nitro phenyl ester by using the F-moc moiety or a photo sensitivenitroatryloxycarbonyl group by solid phase synthesis.

The complexed RNA of the present invention further comprises at leastone RNA (molecule) suitable for transfection purposes, wherein this atleast one RNA (molecule) is complexed with one or more oligopeptides, asdisclosed above with empirical formula I((Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x)).

The at least one RNA (molecule) of the complexed RNA of the presentinvention may have any length (preferably dependent on the type of RNAto be applied as a complexed RNA according to the present invention).Without being restricted thereto, the at least one RNA (molecule) mayhave a length of 5 to 20000 nucleotides, more preferably a length of 5to 10000 or of 300 to 10000 nucleotides, even more preferably a lengthof 5 to 5000 nucleotides, and most preferably a length of 20 to 5000, of50 to 5000, of 100 to 5000 or of 300 to 10000 nucleotides depending onthe type of RNA to be transfected (see disclosure below).

The at least one RNA (molecule) of the complexed RNA of the presentinvention may be any RNA, preferably, without being limited thereto, ashort RNA oligonucleotide (preferable length 5 to 80 or, more preferably20 to 80 nucleotides), a coding RNA, an immunostimulatory RNA, a siRNA,an antisense RNA, or riboswitches, ribozymes or aptamers. Furthermore,the at least one RNA (molecule) of the complexed RNA of the presentinvention may be a single- or a double-stranded RNA (which may also beregarded as an RNA (molecule) due to non-covalent association of twosingle-stranded RNA (molecules)) or a partially double-stranded RNA(which is typically formed by a longer and a shorter single-stranded RNAmolecule or by two single stranded RNA-molecules, which are about equalin length, wherein one single-stranded RNA molecule is partiallycomplementary to the other single-stranded RNA molecule and both thusform a double-stranded RNA molecule in this region). Preferably, the atleast one RNA (molecule) of the complexed RNA of the present inventionmay be a single-stranded RNA. The at least one RNA (molecule) of thecomplexed RNA of the present invention may also be a circular or linearRNA, preferably a linear RNA. More preferably, the at least one RNA(molecule) of the complexed RNA of the present invention may be a(linear) single-stranded RNA. The at least one RNA (molecule) of thecomplexed RNA of the present invention may be a ribosomal RNA (rRNA), atransfer RNA (tRNA), a messenger RNA (mRNA), or a viral RNA (vRNA),preferably a mRNA. The present invention allows all of these RNAs to betransfected into the cell. In this context, an mRNA is typically an RNA,which is composed of several structural elements, e.g. an optional5′-UTR region, an upstream positioned ribosomal binding site followed bya coding region, an optional 3′-UTR region, which may be followed by apoly-A tail (and/or a poly-C-tail). An mRNA may occur as a mono-, di-,or even multicistronic RNA, i.e. an RNA which carries the codingsequences of one, two or more proteins. Such coding sequences in di-, oreven multicistronic mRNA may be separated by at least one IRES sequence,e.g. as defined herein.

Short RNA Oligonucleotides

In a first embodiment, the at least one RNA (molecule) of the complexedRNA of the present invention may be a short RNA oligonucleotide. ShortRNA oligonucleotides in the context of the present invention maycomprise any RNA as defined above. Preferably, the short RNAoligonucleotide may be a single- or a double-stranded RNAoligonucleotide, more preferably a single-stranded RNA oligonucleotide.Even more preferably, the short RNA oligonucleotide may be a linearsingle-stranded RNA oligonucleotide.

Preferably, the short RNA oligonucleotides as used herein comprise alength as defined above in general for RNA molecules, more preferably alength of 5 to 100, of 5 to 50, or of of 30, or, alternatively, a lengthof 20 to 100, of 20 to 80, or, even more preferably, of 20 of 60nucleotides. Short RNA oligonucleotides may be used for variouspurposes, e.g. for (unspecific) immune stimulation, orreducing/suppressing transcription/translation of genes.

Coding RNA

In a second embodiment, the at least one RNA (molecule) of the complexedRNA of the present invention may be a coding RNA. The coding RNA of thecomplexed RNA of the present invention may be any RNA as defined above.Preferably, the coding RNA may be a single- or a double-stranded RNA,more preferably a single-stranded RNA, and/or a circular or linear RNA,more preferably a linear RNA. Even more preferably, the coding RNA maybe a (linear) single-stranded RNA. Most preferably, the coding RNA maybe a ((linear) single-stranded) messenger RNA (mRNA).

The coding RNA may further encode a protein or a peptide, which may beselected, without being restricted thereto, e.g. from therapeuticallyactive proteins or peptides, tumor antigens, antibodies,immunostimulating proteins or peptides, etc., or from any other proteinor peptide suitable for a specific (therapeutic) application, whereinthe at least one RNA (molecule) encoding the protein is to betransported into a cell, a tissue or an organism and the protein isexpressed subsequently in this cell, tissue or organism.

In this context, therapeutically active proteins may be selected fromany recombinant or isolated proteins known to a skilled person from theprior art. Without being restricted thereto therapeutically activeproteins as encoded by the at least one RNA (molecule) of the complexedRNA as defined herein may be selected from apoptotic factors orapoptosis related proteins including AIF, Apaf e.g. Apaf-1, Apaf-2,Apaf-3, oder APO-2 (L), APO-3 (L), Apopain, Bad, Bak, Bax, Bcl-2,Bcl-x_(L), Bcl-x_(S), bik, CAD, Calpain, Caspase e.g. Caspase-1,Caspase-2, Caspase-3, Caspase-4, Caspase-5, Caspase-6, Caspase-7,Caspase-8, Caspase-9, Caspase-10, Caspase-11, ced-3, ced-9, c-jun,c-Myc, crm A, cytochrom C, CdR1, DcR1, DD, DED, DISC, DNA-PK_(CS), DR3,DR4, DR5, FADD/MORT-1, FAK, Fas (Fas-ligand CD95/fas (receptor)),FLICE/MACH, FLIP, fodrin, fos, G-Actin, Gas-2, gelsolin, granzyme A/B,ICAD, ICE, JNK, lamin A/B, MAP, MCL-1, Mdm-2, MEKK-1, MORT-1, NEDD,NF-_(kappa)B, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKCdelta, pRb,presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidinkinasefrom herpes simplex, TRADD, TRAF2, TRAIL-R1, TRAIL-R2, TRAIL-R3,transglutaminase, etc.

Therapeutically active proteins as encoded by the at least one RNA(molecule) of the complexed RNA as defined herein may also be selectedfrom recombinant proteins, including proteins selected from the groupconsisting of 0ATL3, 0FC3, 0PA3, 0PD2, 4-1BBL, 5T4, 6Ckine, 707-AP, 9D7,A2M, AA, AAAS, AACT, AASS, ABAT, ABCA1, ABCA4, ABCB1, ABCB11, ABCB2,ABCB4, ABCB7, ABCC2, ABCC6, ABCC8, ABCD1, ABCD3, ABCG5, ABCG8, ABL1,ABO, ABR ACAA1, ACACA, ACADL, ACADM, ACADS, ACADVL, ACAT1, ACCPN, ACE,ACHE, ACHM3, ACHM1, ACLS, ACPI, ACTA1, ACTC, ACTN4, ACVRL1, AD2, ADA,ADAMTS13, ADAMTS2, ADFN, ADH1B, ADH1C, ADLDH3A2, ADRB2, ADRB3, ADSL,AEZ, AFA, AFD1, AFP, AGA, AGL, AGMX2, AGPS, AGS1, AGT, AGTR1, AGXT,AH02, AHCY, AHDS, AHHR, AHSG, AIC, AIED, AIH2, AIH3, AIM-2, AIPL1, AIRE,AK1, ALAD, ALAS2, ALB, HPG1, ALDH2, ALDH3A2, ALDH4A1, ALDH5A1, ALDH1A1,ALDOA, ALDOB, ALMS1, ALPL, ALPP, ALS2, ALX4, AMACR, AMBP, AMCD, AMCD1,AMCN, AMELX, AMELY, AMGL, AMH, AMHR2, AMPD3, AMPD1, AMT, ANC, ANCR,ANK1, ANOP1, AOM, AP0A4, AP0C2, AP0C3, AP3B1, APC, aPKC, APOA2, APOA1,APOB, APOC3, APOC2, APOE, APOH, APP, APRT, APS1, AQP2, AR, ARAF1, ARG1,ARHGEF12, ARMET, ARSA, ARSB, ARSC2, ARSE, ART-4, ARTC1/m, ARTS, ARVD1,ARX, AS, ASAH, ASAT, ASD1, ASL, ASMD, ASMT, ASNS, ASPA, ASS, ASSP2,ASSP5, ASSP6, AT3, ATD, ATHS, ATM, ATP2A1, ATP2A2, ATP2C1, ATP6B1,ATP7A, ATP7B, ATP8B1, ATPSK2, ATRX, ATXN1, ATXN2, ATXN3, AUTS1, AVMD,AVP, AVPR2, AVSD1, AXIN1, AXIN2, AZF2, B2M, B4GALT7, B7H4, BAGE, BAGE-1,BAX, BBS2, BBS3, BBS4, BCA225, BCAA, BCH, BCHE, BCKDHA, BCKDHB, BCL10,BCL2, BCL3, BCL5, BCL6, BCPM, BCR, BCR/ABL, BDC, BDE, BDMF, BDMR, BEST1,beta-Catenin/m, BF, BFHD, BFIC, BFLS, BFSP2, BGLAP, BGN, BHD, BHR1,BING-4, BIRC5, BJS, BLM, BLMH, BLNK, BMPR2, BPGM, BRAF, BRCA1, BRCA1/m,BRCA2, BRCA2/m, BRCD2, BRCD1, BRDT, BSCL, BSCL2, BTAA, BTD, BTK, BUB1,BWS, BZX, C0L2A1, C0L6A1, C1NH, C1QA, C1QB, C1QG, C1S, C2, C3, C4A, C4B,C5, C6, C7, C7orf2, C8A, C8B, C9, CA125, CA15-3/CA 27-29, CA195, CA19-9,CA72-4, CA2, CA242, CA50, CABYR, CACD, CACNA2D1, CACNA1A, CACNA1F,CACNA1S, CACNB2, CACNB4, CAGE, CA1, CALB3, CALCA, CALCR, CALM, CALR,CAM43, CAMEL, CAP-1, CAPN3, CARD15, CASP-5/m, CASP-8, CASP-8/m, CASR,CAT, CATM, CAV3, CB1, CBBM, CBS, CCA1, CCAL2, CCAL1, CCAT, CCL-1,CCL-11, CCL-12, CCL-13, CCL-14, CCL-15, CCL-16, CCL-17, CCL-18, CCL-19,CCL-2, CCL-20, CCL-21, CCL-22, CCL-23, CCL-24, CCL-25, CCL-27, CCL-3,CCL-4, CCL-5, CCL-7, CCL-8, CCM1, CCNB1, CCND1, CCO, CCR2, CCR5, CCT,CCV, CCZS, CD1, CD19, CD20, CD22, CD25, CD27, CD27L, cD3, CD30, CD30,CD30L, CD33, CD36, CD3E, CD3G, CD3Z, CD4, CD40, CD40L, CD44, CD44v,CD44v6, CD52, CD55, CD56, CD59, CD80, CD86, CDAN1, CDAN2, CDAN3, CDC27,CDC27/m, CDC2L1, CDH1, CDK4, CDK4/m, CDKN1C, CDKN2A, CDKN2A/m, CDKN1A,CDKN1C, CDL1, CDPD1, CDR1, CEA, CEACAM1, CEACAM5, CECR, CECR9, CEPA,CETP, CFNS, CFTR, CGF1, CHAC, CHED2, CHED1, CHEK2, CHM, CHML, CHR39C,CHRNA4, CHRNA1, CHRNB1, CHRNE, CHS, CHS1, CHST6, CHX10, CIAS1, CIDX,CKN1, CLA2, CLA3, CLA1, CLCA2, CLCN1, CLCN5, CLCNKB, CLDN16, CLP, CLN2,CLN3, CLN4, CLN5, CLN6, CLN8, C1QA, C1QB, C1QG, C1R, CLS, CMCWTD, CMDJ,CMD1A, CMD1B, CMH2, MH3, CMH6, CMKBR2, CMKBR5, CML28, CML66, CMM, CMT2B,CMT2D, CMT4A, CMT1A, CMTX2, CMTX3, C-MYC, CNA1, CND, CNGA3, CNGA1,CNGB3, CNSN, CNTF, COA-1/m, COCH, COD2, COD1, COH1, COL10A, COL2A2,COL11A2, COL17A1, COL1A1, COL1A2, COL2A1, COL3A1, COL4A3, COL4A4,COL4A5, COL4A6, COL5A1, COL5A2, COL6A1, COL6A2, COL6A3, COL7A1, COL8A2,COL9A2, COL9A3, COL11A1, COL1A2, COL23A1, COL1A1, COLQ, COMP, COMT,CORD5, CORD1, COX10, COX-2, CP, CPB2, CPO, CPP, CPS1, CPT2, CPT1A, CPX,CRAT, CRB1, CRBM, CREBBP, CRH, CRHBP, CRS, CRV, CRX, CRYAB, CRYBA1,CRYBB2, CRYGA, CRYGC, CRYGD, CSA, CSE, CSF1R, CSF2RA, CSF2RB, CSF3R,CSF1R, CST3, CSTB, CT, CT7, CT-9/BRD6, CTAA1, CTACK, CTEN, CTH, CTHM,CTLA4, CTM, CTNNB1, CTNS, CTPA, CTSB, CTSC, CTSK, CTSL, CTS1, CUBN,CVD1, CX3CL1, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL16, CXCL2,CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CYB5, CYBA, CYBB,CYBB5, CYFRA 21-1, CYLD, CYLD1, CYMD, CYP11B1, CYP11B2, CYP17, CYP17A1,CYP19, CYP19A1, CYP1A2, CYP1B1, CYP21A2, CYP27A1, CYP27B1, CYP2A6,CYP2C, CYP2C19, CYP2C9, CYP2D, CYP2D6, CYP2D7P1, CYP3A4, CYP7B1, CYPB1,CYP11B1, CYP1A1, CYP1B1, CYRAA, D40, DADI, DAM, DAM-10/MAGE-B1,DAM-6/MAGE-B2, DAX1, DAZ, DBA, DBH, DBI, DBT, DCC, DC-CK1, DCK, DCR,DCX, DDB 1, DDB2, DDIT3, DDU, DECR1, DEK-CAN, DEM, DES, DF, DFN2, DFN4,DFN6, DFNA4, DFNA5, DFNB5, DGCR, DHCR7, DHFR, DHOF, DHS, DIA1, DIAPH2,DIAPH1, DIH1, DIO1, DISCI, DKC1, DLAT, DLD, DLL3, DLX3, DMBT1, DMD, DM1,DMPK, DMWD, DNAI1, DNASE1, DNMT3B, DPEP1, DPYD, DPYS, DRD2, DRD4, DRPLA,DSCR1, DSG1, DSP, DSPP, DSS, DTDP2, DTR, DURS1, DWS, DYS, DYSF, DYT2,DYT3, DYT4, DYT2, DYT1, DYX1, EBAF, EBM, EBNA, EBP, EBR3, EBS1, ECA1,ECB2, ECE1, ECGF1, ECT, ED2, ED4, EDA, EDAR, ECA1, EDN3, EDNRB, EEC1,EEF1A1L14, EEGV1, EFEMP1, EFTUD2/m, EGFR, EGFR/Her1, EGI, EGR2, EIF2AK3,eIF4G, EKV, EI IS, ELA2, ELF2, ELF2M, ELK1, ELN, ELONG, EMD, EML1,EMMPRIN, EMX2, ENA-78, ENAM, END3, ENG, ENO1, ENPP1, ENUR2, ENUR1, EOS,EP300, EPB41, EPB42, EPCAM, EPD, EphA1, EphA2, EphA3, EphrinA2,EphrinA3, EPHX1, EPM2A, EPO, EPOR, EPX, ERBB2, ERCC2 ERCC3, ERCC4,ERCC5, ERCC6, ERVR, ESR1, ETFA, ETFB, ETFDH, ETM1, ETV6-AML1, ETV1, EVC,EVR2, EVR1, EWSR1, EXT2, EXT3, EXT1, EYA1, EYCL2, EYCL3, EYCL1, EZH2,F10, F11, F12, F13A1, F13B, F2, F5, F5F8D, F7, F8, F8C, F9, FABP2,FACL6, FAH, FANCA, FANCB, FANCC, FANCD2, FANCF, FasL, FBN2, FBN1, FBP1,FCG3RA, FCGR2A, FCGR2B, FCGR3A, FCHL, FCMD, FCP1, FDPSL5, FECH, FEO,FEOM1, FES, FGA, FGB, FGD1, FGF2, FGF23, FGF5, FGFR2, FGFR3, FGFR1, FGG,FGS1, FH, FIC1, FIH, F2, FKBP6, FLNA, FLT4, FMO3, FMO4, FMR2, FMR1, FN,FN1/m, FOXC1, FOXE1, FOXL2, FOXO1A, FPDMM, FPF, Fra-1, FRAXF, FRDA,FSHB, FSHMD1A, FSHR, FTH1, FTHL17, FTL, FTZF1, FUCA1, FUT2, FUT6, FUT1,FY, G250, G250/CAIX, G6PC, G6PD, G6PT1, G6PT2, GAA, GABRA3, GAGE-1,GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7b, GAGE-8, GALC, GALE,GALK1, GALNS, GALT, GAMT, GAN, GAST, GASTRIN17, GATA3, GATA, GBA, GBE,GC, GCDH, GCGR, GCH1, GCK, GCP-2, GCS1, G-CSF, GCSH, GCSL, GCY, GDEP,GDF5, GDI1, GDNF, GDXY, GFAP, GFND, GGCX, GGT1, GH2, GH1, GHR, GHRHR,GHS, GIF, GINGF, GIP, GJA3, GJA8, GJB2, GJB3, GJB6, GJB1, GK, GLA, GLB,GLB1, GLC3B, GLC1B, GLC1C, GLDC, GLI3, GLP1, GLRA1, GLUD1, GM1(fuc-GM1), GM2A, GM-CSF, GMPR, GNAI2, GNAS, GNAT1, GNB3, GNE, GNPTA,GNRH, GNRH1, GNRHR, GNS, GnT-V, gp100, GP1BA, GP1BB, GP9, GPC3, GPD2,GPDS1, GP1, GP1BA, GPN1LW, GPNMB/m, GPSC, GPX1, GRHPR, GRK1, GRO, GRO,GRO, GRPR, GSE, GSM1, GSN, GSR, GSS, GTD, GTS, GUCAIA, GUCY2D, GULOP,GUSB, GUSM, GUST, GYPA, GYPC, GYS1, GYS2, HOKPP2, HOMG2, HADHA, HADHB,HAGE, HAGH, HAL, HAST-2, HB 1, HBA2, HBA1, HBB, HBBP1, HBD, HBE1, HBG2,HBG1, HBHR, HBP1, HBQ1, HBZ, HBZP, HCA, HCC-1, HCC-4, HCF2, HCG, HCL2,HCL1, HCR, HCVS, HD, HPN, HER2, HER2/NEU, HER3, HERV-K-MEL, HESX1, HEXA,HEXB, HF1, HFE, HF1, HGD, HHC2, HHC3, HHG, HK1 HLA-A, HLA-A*0201-R1701,HLA-A11/m, HLA-A2/m, HLA-DPB1 HLA-DRA, HLCS, HLXB9, HMBS, HMGA2, HMGCL,HMI, HMN2, HMOX1, HMS1 HMW-MAA, HND, HNE, HNF4A, HOAC, HOMEOBOX NKX 3.1,HOM-TES-14/SCP-1, HOM-TES-85, HOXA1 HOXD13, HP, HPC1, HPD, HPE2, HPE1,HPFH, HPFH2, HPRT1, HPS1, HPT, HPV-E6, HPV-E7, HR, HRAS, HRD, HRG,HRPT2, HRPT1, HRX, HSD118B2, HSD17B3, HSD17B4, HSD3B2, HSD3B3, HSN1,HSP70-2M, HSPG2, HST-2, HTC2, HTC1, hTERT, HTN3, HTR2C, HVBS6, HVBS1,HVEC, HV1S, HYAL1, HYR, 1-309, IAB, IBGC1, IBM2, ICAM1, ICAM3, iCE,ICHQ, ICR5, ICR1, ICS 1, IDDM2, IDDM1, IDS, IDUA, IF, IFNa/b, IFNGR1,IGAD1, IGER, IGF-1R, IGF2R, IGF1, IGH, IGHC, IGHG2, IGHG1, IGHM, IGHR,IGKC, IHG1, IHH, IKBKG, IL1, IL-1 RA, IL10, IL-11, IL12, IL12RB1, IL13,IL-13Rα2, IL-15, IL-16, IL-17, IL18, IL-1a, IL-1α, IL-1b, IL-1β,IL1RAPL1, IL2, IL24, IL-2R, IL2RA, IL2RG, IL3, IL3RA, IL4, IL4R, IL4R,IL-5, IL6, IL-7, IL7R, IL-8, IL-9, Immature laminin receptor, IMMP2L,INDX, INFGR1, INFGR2, INFα, IFNβINFγ, INS, INSR, INVS, IP-10, IP2, IPF1,IP1, IRF6, IRS1, ISCW, ITGA2, ITGA2B, ITGA6, ITGA7, ITGB2, ITGB3, ITGB4,ITIH1, ITM2B, IV, IVD, JAG1, JAK3, JBS, JBTS1, JMS, JPD, KAL1, KAL2,KAL1, KLK2, KLK4, KCNA1, KCNE2, KCNE1, KCNH2, KCNJ1, KCNJ2, KCNJ1,KCNQ2, KCNQ3, KCNQ4, KCNQ1, KCS, KERA, KFM, KFS, KFSD, KHK, ki-67,KIAA0020, KIAA0205, KIAA0205/m, KIF B, KIT, KK-LC-1, KLK3, KLKB1,KM-HN-1, KMS, KNG, KNO, K-RAS/m, KRAS2, KREV1, KRT1, KRT10, KRT12,KRT13, KRT14, KRT14L1, KRT14L2, KRT14L3, KRT16, KRT16L1, KRT16L2, KRT17,KRT18, KRT2A, KRT3, KRT4, KRT5, KRT6 A, KRT6B, KRT9, KRTHB1, KRTHB6,KRT1, KSA, KSS, KWE, KYNU, LOH19CR1, L1CAM, LAGE, LAGE-1, LALL, LAMA2,LAMA3, LAMB3, LAMB1, LAMC2, LAMP2, LAP, LCA5, LCAT, LCCS, LCCS 1, LCFS2,LCS1, LCT, LDHA, LDHB, LDHC, LDLR, LDLR/FUT, LEP, LEWISY, LGCR,LGGF-PBP, LGI1, LGMD2H, LGMD1A, LGMD1B, LHB, LHCGR, LHON, LHRH, LHX3,LIF, LIG1, LIMM, LIMP2, LIPA, LIPA, LIPB, LIPC, LIVIN, L1CAM, LMAN1,LMNA, LMX1B, LOLR, LOR, LOX, LPA, LPL, LPP, LQT4, LRP5, LRS 1, LSFC,LT-β, LTBP2, LTC4S, LYL1, XCL1, LYZ, M344, MA50, MAA, MADH4, MAFD2,MAFD1, MAGE, MAGE-A1, MAGE-A10, MAGE-A12, MAGE-A2, MAGE-A3, MAGE-A4,MAGE-A6, MAGE-A9, MAGEB1, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-B2,MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1,MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, MGB1,MGB2, MAN2A1, MAN2B1, MANBA, MANBB, MAOA, MAOB, MAPK81P1, MAPT, MART-1,MART-2, MART2/m, MAT1A, MBL2, MBP, MBS1, MC1R, MC2R, MC4R, MCC, MCCC2,MCCC1, MCDR1, MCF2, MCKD, MCL1, MC1R, MCOLN1, MCOP, MCOR, MCP-1, MCP-2,MCP-3, MCP-4, MCPH2, MCPH1, MCS, M-CSF, MDB, MDCR, MDM2, MDRV, MDS 1,ME1, ME1/m, ME2, ME20, ME3, MEAX, MEB, MEC CCL-28, MECP2, MEFV, MELANA,MELAS, MEN1 MSLN, MET, MF4, MG50, MG50/PXDN, MGAT2, MGAT5, MGC1 MGCR,MGCT, MGI, MGP, MHC2TA, MHS2, MHS4, MIC2, MIC5, MIDI, MIF, MIP,MIP-5/HCC-2, MITF, MJD, MK167, MKKS, MKS1, MLH1, MLL, MLLT2, MLLT3,MLLT7, MLLT1, MLS, MLYCD, MMA1a, MMP 11, MMVP1, MN/CA IX-Antigen, MNG1,MN1, MOC31, MOCS2, MOCS1, MOG, MORC, MOS, MOV18, MPD1, MPE, MPFD, MPI,MPIF-1, MPL, MPO, MPS3C, MPZ, MRE11A, MROS, MRP1, MRP2, MRP3, MRSD,MRX14, MRX2, MRX20, MRX3, MRX40, MRXA, MRX1, MS, MS4A2, MSD, MSH2, MSH3,MSH6, MSS, MSSE, MSX2, MSX1, MTATP6, MTC03, MTCO1, MTCYB, MTHFR, MTM1,MTMR2, MTND2, MTND4, MTND5, MTND6, MTND1, MTP, MTR, MTRNR2, MTRNR1,MTRR, MTTE, MTTG, MTTI, MTTK, MTTL2, MTTL1, MTTN, MTTP, MTTS1, MUC1,MUC2, MUC4, MUCSAC, MUM-1, MUM-1/m, MUM-2, MUM-2/m, MUM-3, MUM-3/m, MUT,mutant p21 ras, MUTYH, MVK, MX2, MXI1, MYOS5A, MYB, MYBPC3, MYC, MYCL2,MYH6, MYH7, MYL2, MYL3, MYMY, MYO15A, MYO1G, MYOSA, MYO7A, MYOC,Myosin/m, MYP2, MYP1, NA88-A, N-acetylglucosaminyltransferase-V, NAGA,NAGLU, NAMSD, NAPB, NAT2, NAT, NBIA1, NBS1, NCAM, NCF2, NCF1, NDN, NDP,NDUFS4, NDUFS7, NDUFS8, NDUFV1, NDUFV2, NEB, NEFH, NEM1, Neo-PAP,neo-PAP/m, NEU1, NEUROD1, NF2, NF1, NFYC/m, NGEP, NHS, NKS1, NKX2E, NM,NME1, NMP22, NMTC, NODAL, NOG, NOS3, NOTCH3, NOTCH1, NP, NPC2, NPC1,NPHL2, NPHP1, NPHS2, NPHS1, NPM/ALK, NPPA, NQO1, NR2E3, NR3C1, NR3C2,NRAS, NRAS/m, NRL, NROB1, NRTN, NSE, NSX, NTRK1, NUMA1, NXF2, NY-CO1,NY-ESO1, NY-ESO-B, NY-LU-12, ALDOA, NYS2, NYS4, NY-SAR-35, NYS1, NYX,OA3, OA1, OAP, OASD, OAT, OCA1, OCA2, OCD1, OCRL, OCRL1, OCT, ODDD,ODT1, OFC1, OFD1, OGDH, OGT, OGT/m, OPA2, OPA1, OPD1, OPEM, OPG, OPN,OPN1LW, OPN1MW, OPN1SW, OPPG, OPTB1, TTD, ORM1, ORP1, OS-9, OS-9/m, OSMLIF, OTC, OTOF, OTSC1, OXCT1, OYTES1, P15, P190 MINOR BCR-ABL, P2RY12,P3, P16, P40, P4HB, P-501, P53, P53/m, P97, PABPN1, PAFAHIB1, PAFAH1P1,PAGE-4, PAGE-5, PAH, PAI-1, PAI-2, PAK3, PAP, PAPPA, PARK2, PART-1,PATE, PAX2, PAX3, PAX6, PAX7, PAX8, PAX9, PBCA, PBCRA1, PBT, PBX1,PBXP1, PC, PCBD, PCCA, PCCB, PCK2, PCK1, PCLD, PCOS1, PCSK1, PDB1, PDCN,PDE6A, PDE6B, PDEF, PDGFB, PDGFR, PDGFRL, PDHA1, PDR, PDX1, PECAM1,PEE1, PEO1, PEPD, PEX10, PEX12, PEX13, PEX3, PEX5, PEX6, PEX7, PEX1,PF4, PFB1, PFC, PFKFB, PFKM, PGAM2, PGD, PGK1, PGK1P1, PGL2, PGR, PGS,PHA2A, PHB, PHEX, PHGDH, PHKA2, PHKA1, PHKB, PHKG2, PHP, PHYH, PI, PI3,PIGA, PIM1-KINASE, PIN1, PIP5K1B, PITX2, PITX3, PKD2, PKD3, PKD1, PKDTS,PKHD1, PKLR, PKP1, PKU1, PLA2G2A, PLA2G7, PLAT, PLEC1, PLG, PLI, PLOD,PLP1, PMEL17, PML, PML/RARα, PMM2, PMP22, PMS2, PMS1, PNKD, PNLIP, POF1,POLA, POLH, POMC, PON2, PON1, PORC, POTE, POU1F1, POU3F4, POU4F3,POU1F1, PPAC, PPARG, PPCD, PPGB, PPH1, PPKB, PPMX, PPOX, PPPIR3A,PPP2R2B, PPT1, PRAME, PRB, PRB3, PRCA1, PRCC, PRD, PRDX5/m, PRF1, PRG4,PRKAR1A, PRKCA, PRKDC, PRKWNK4, PRNP, PROC, PRODH, PROM1, PROP1, PROS1,PRST, PRP8, PRPF31, PRPF8, PRPH2, PRPS2, PRPS1, PRS, PRSS7, PRSS1,PRTN3, PRX, PSA, PSAP, PSCA, PSEN2, PSEN1, PSG1, PSGR, PSM, PSMA,PSORS1, PTC, PTCH, PTCH1, PTCH2, PTEN, PTGS1, PTH, PTHR1, PTLAH, PTOS1,PTPN12, PTPNI I, PTPRK, PTPRK/m, PTS, PUJO, PVR, PVRL1, PWCR, PXE,PXMP3, PXR1, PYGL, PYGM, QDPR, RAB27A, RAD54B, RAD54L, RAG2, RAGE,RAGE-1, RAG1, RAP1, RARA, RASA1, RBAF600/m, RB1, RBP4, RBP4, RBS, RCA1,RCAS1, RCCP2, RCD1, RCV1, RDH5, RDPA, RDS, RECQL2, RECQL3, RECQL4,REG1A, REHOBE, REN, RENBP, RENS1, RET, RFX5, RFXANK, RFXAP, RGR, RHAG,RHAMM/CD168, RHD, RHO, Rip-1, RLBP1, RLN2, RLN1, RLS, RMD1, RMRP, ROM1,ROR2, RP, RP1, RP14, RP17, RP2, RP6, RP9, RPD1, RPE65, RPGR, RPGRIP1,RP1, RP10, RPS19, RPS2, RPS4X, RPS4Y, RPS6KA3, RRAS2, RS1, RSN, RSS,RU1, RU2, RUNX2, RUNX1, RWS, RYR1, S-100, SAA1, SACS, SAG, SAGE, SALL1,SARDH, SART1, SART2, SART3, SAS, SAX1, SCA2, SCA4, SCA5, SCA7, SCA8,SCA1, SCC, SCCD, SCF, SCLC1, SCN1A, SCN1B, SCN4A, SCN5A, SCNN1A, SCNN1B,SCNN1G, SCO2, SCP1, SCZD2, SCZD3, SCZD4, SCZD6, SCZD1, SDF-1α/β SDHA,SDHD, SDYS, SEDL, SERPENA7, SERPINA3, SERPINA6, SERPINA1, SERPINC1,SERPIND1, SERPINE1, SERPINF2, SERPING1, SERPINI1, SFTPA1, SFTPB, SFTPC,SFTPD, SGCA, SGCB, SGCD, SGCE, SGM1, SGSH, SGY-1, SH2D1A, SHBG, SHFM2,SHFM3, SHFM1, SHH, SHOX, SI, SIAL, SIALYL LEWISX, SIASD, S11, SIM1,SIRT2/m, SIX3, SJS1, SKP2, SLC10A2, SLC12A1, SLC12A3, SLC17A5, SLC19A2,SLC22A1L, SLC22A5, SLC25A13, SLC25A15, SLC25A20, SLC25A4, SLC25A5,SLC25A6, SLC26A2, SLC26A3, SLC26A4, SLC2A1, SLC2A2, SLC2A4, SLC3A1,SLC4A1, SLC4A4, SLC5A1, SLC5A5, SLC6A2, SLC6A3, SLC6A4, SLC7A7, SLC7A9,SLC11A1, SLOS, SMA, SMAD1, SMAL, SMARCB1, SMAX2, SMCR, SMCY, SM1, SMN2,SMN1, SMPD1, SNCA, SNRPN, SOD2, SOD3, SOD1, SOS1, SOST, SOX9, SOX10,Sp17, SPANXC, SPG23, SPG3A, SPG4, SPG5A, SPG5B, SPG6, SPG7, SPINK1,SPINK5, SPPK, SPPM, SPSMA, SPTA1, SPTB, SPTLC1, SRC, SRD5A2, SRPX, SRS,SRY, βhCG, SSTR2, SSX1, SSX2 (HOM-MEL-40/SSX2), SSX4, ST8, STAMP-1,STAR, STARP1, STATH, STEAP, STK2, STK11, STn/KLH, STO, STOM, STS, SUOX,SURF1, SURVIVIN-2B, SYCP1, SYM1, SYN1, SYNS1, SYP, SYT/SSX, SYT-SSX-1,SYT-SSX-2, TA-90, TAAL6, TACSTD1, TACSTD2, TAG72, TAF7L, TAF1, TAGE,TAG-72, TALI, TAM, TAP2, TAP1, TAPVR1, TARC, TARP, TAT, TAZ, TBP, TBX22,TBX3, TBX5, TBXA2R, TBXAS1, TCAP, TCF2, TCF1, TCIRG1, TCL2, TCL4, TCL1A,TCN2, TCOF1, TCR, TCRA, TDD, TDFA, TDRD1, TECK, TECTA, TEK, TEL/AML1,TELAB1, TEX15, TF, TFAP2B, TFE3, TFR2, TG, TGFα, TGFβ, TGFμ1, TGFβ1,TGFβR2, TGFβRE, TGFγ, TGFβRII, TGIF, TGM-4, TGM1, TH, THAS, THBD, THC,THC2, THM, THPO, THRA, THRB, TIMM8A, TIMP2, TIMP3, TIMP1, TITF1, TKCR,TKT, TLP, TLR1, TLR10, TLR2, TLR3, TLR4, TLR4, TLR5, TLR6, TLR7, TLR8,TLR9, TLX1, TM4SF1, TM4SF2, TMC1, TMD, TMIP, TNDM, TNF, TNFRSF11A,TNFRSF1A, TNFRSF6, TNFSF5, TNFSF6, TNFα, TNFβ, TNNI3, TNNT2, TOC, TOP2A,TOP1, TP53, TP63, TPA, TPBG, TPI, TPI/m, TPI1, TPM3, TPM1, TPMT, TPO,TPS, TPTA, TRA, TRAG3, TRAPPC2, TRC8, TREH, TRG, TRH, TRIM32, TRIM37,TRP1, TRP2, TRP-2/6b, TRP-2/INT2, Trp-p8, TRPS1, TS, TSC2, TSC3, TSC1,TSG101, TSHB, TSHR, TSP-180, TST, TTGA2B, TTN, TTPA, TTR, TU M2-PK,TULP1, TWIST, TYH, TYR, TYROBP, TYROBP, TYRP1, TYS, UBE2A, UBE3A, UBE1,UCHL1, UFS, UGT1A, ULR, UMPK, UMPS, UOX, UPA, UQCRC1, URO5, UROD, UPK1B,UROS, USH2A, USH3A, USH1A, USH1C, USP9Y, UV24, VBCH, VCF, VDI, VDR,VEGF, VEGFR-2, VEGFR-1, VEGFR-2/FLK-1, VHL, VIM, VMD2, VMD1, VMGLOM,VNEZ, VNF, VP, VRNI, VWF, VWS, WAS, WBS2, WFS2, WFS1, WHCR, WHN, WISP3,WMS, WRN, WS2A, WS2B, WSN, WSS, WT2, WT3, WT1, WTS, WWS, XAGE, XDH, XIC,XIST, XK, XM, XPA, XPC, XRCC9, XS, ZAP70, ZFHX1B, ZFX, ZFY, ZIC2, ZIC3,ZNF145, ZNF261, ZNF35, ZNF41, ZNF6, ZNF198, and ZWS1.

Additionally, therapeutically active proteins as encoded by the at leastone RNA (molecule) of the complexed RNA as defined herein may also beselected from growth hormones or growth factors, for example forpromoting growth in a (transgenic) living being, such as, for example,TGFα and the IGFs (insulin-like growth factors), proteins that influencethe metabolism and/or haematopoiesis, such as, for example,α-anti-trypsin, LDL receptor, erythropoietin (EPO), insulin, GATA-1,etc., or proteins such as, for example, factors VIII and XI of the bloodcoagulation system, etc. Such proteins further include enzymes, such as,for example, β-galactosidase (lacZ), DNA restriction enzymes (e.g.EcoRI, HindIII, etc.), lysozymes, etc., or proteases, such as, forexample, papain, bromelain, keratinases, trypsin, chymotrypsin, pepsin,renin (chymosin), suizyme, nortase, etc. These proteins may be encodedby the at least one RNA (molecule) of the complexed RNA as definedherein. Accordingly, the invention provides a technology which allows tosubstitute proteins which are defective in the organism to be treated(e.g. either due to mutations, due to defective or missing expression)and thereby effective and increased expression of proteins, which arenot functional in the organism to be treated, as e.g. occurring inmonogenetic disorders, preferably without leading to an innate immuneresponse.

Alternatively, therapeutically active proteins as encoded by the atleast one RNA (molecule) of the complexed RNA as defined herein may alsobe selected from proteases etc. which allow to cure a specific diseasedue to e.g. (over)expression of a dysfunctional or exogenous proteinscausing disorders or diseases. Accordingly, the invention may be used totherapeutically introduce the complexed RNA into the organism, whichattacks a pathogenic organism (virus, bacteria etc). E.g. RNA encodingtherapeutic proteases may be used to cleave viral proteins which areessential to the viral assembly or other essential steps of virusproduction.

Therapeutically active proteins as encoded by the at least one RNA(molecule) of the complexed RNA as defined herein may also be selectedfrom proteins which modulate various intracellular pathways by e.g.signal transmission modulation (inhibition or stimulation) which mayinfluence pivotal intracellular processes like apoptosis, cell growthetc, in particular with respect to the organism's immune system.Accordingly, immune modulators, e.g. cytokines, lymphokines, monokines,interferones etc. may be expressed efficiently by the complexed RNA asdefined herein. Preferably, these proteins therefore also include, forexample, cytokines of class I of the cytokine family that contain 4position-specific conserved cysteine residues (CCCC) and a conservedsequence motif Trp-Ser-X-Trp-Ser (WSXWS), wherein X represents anunconserved amino acid. Cytokines of class I of the cytokine familyinclude the GM-CSF sub-family, for example IL-3, IL-5, GM-CSF, the IL-6sub-family, for example IL-6, IL-11, IL-12, or the IL-2 sub-family, forexample IL-2, IL-4, IL-7, IL-9, IL-15, etc., or the cytokines IL-1α,IL-1β, IL-10 etc. By analogy, such proteins can also include cytokinesof class II of the cytokine family (interferon receptor family), whichlikewise contain 4 position-specific conserved cysteine residues (CCCC)but no conserved sequence motif Trp-Ser-X-Trp-Ser (WSXWS). Cytokines ofclass II of the cytokine family include, for example, IFN-α, IFN-β,IFN-γ, etc. Proteins coded for by the at least one modified (m)RNA (ofthe inventive immunosuppressive composition) used according to theinvention can further include also cytokines of the tumour necrosisfamily, for example TNF-α, TNF-β, TNF-RI, TNF-RII, CD40, Fas, etc., orcytokines of the chemokine family, which contain 7 transmembrane helicesand interact with G-protein, for example IL-8, MIP-1, RANTES, CCR5,CXR4, etc. Such proteins can also be selected from apoptosis factors orapoptosis-related or -linked proteins, including AIF, Apaf, for exampleApaf-1, Apaf-2, Apaf-3, or APO-2 (L), APO-3 (L), apopain, Bad, Bak, Bax,Bcl-2, Bcl-x_(L), Bcl-x_(S), bik, CAD, calpain, caspases, for examplecaspase-1, caspase-2, caspase-3, caspase-4, caspase-5, caspase-6,caspase-7, caspase-8, caspase-9, caspase-10, caspase-11, ced-3, ced-9,c-Jun, c-Myc, crm A, cytochrome C, CdR1, DcR1, DD, DED, DISC,DNA-PK_(CS), DR3, DR4, DR5, FADD/MORT-1, FAK, Fas (Fas ligand CD95/fas(receptor)), FLICE/MACH, FLIP, fodrin, fos, G-actin, Gas-2, gelsolin,granzymes A/B, ICAD, ICE, JNK, lamin A/B, MAP, MCL-1, Mdm-2, MEKK-1,MORT-1, NEDD, NF-_(κ)B, NuMa, p53, PAK-2, PARP, perforin, PITSLRE, PKCδ,pRb, presenilin, prICE, RAIDD, Ras, RIP, sphingomyelinase, thymidinekinase from Herpes simplex, TRADD, TRAF2, TRAIL, TRAIL-R1, TRAIL-R2,TRAIL-R3, transglutaminase, etc.

Additionally, therapeutically active proteins as encoded by the at leastone RNA (molecule) of the complexed RNA as defined herein may also codefor antigen specific T cell receptors. The T cell receptor or TCR is amolecule found on the surface of T lymphocytes (or T cells) that isgenerally responsible for recognizing antigens bound to majorhistocompatibility complex (MHC) molecules. It is a heterodimerconsisting of an alpha and beta chain in 95% of T cells, while 5% of Tcells have TCRs consisting of gamma and delta chains. Engagement of theTCR with antigen and MHC results in activation of its T lymphocytethrough a series of biochemical events mediated by associated enzymes,co-receptors and specialized accessory molecules. Hence, these proteinsallow to specifically target specific antigen and may support thefunctionality of the immune system due to their targeting properties.Accordingly, transfection of cells in vivo by administering the at leastone RNA (molecule) of the complexed RNA as defined herein coding forthese receptors or, preferably, an ex vivo cell transfection approach(e.g. by transfecting specifically certain immune cells), may bepursued. The T cell receptor molecules introduced recognize specificantigens on MHC molecule and may thereby support the immune system'sawareness of antigens to be attacked.

The therapeutically active proteins, which may be encoded by the atleast one RNA (molecule) of the complexed RNA as defined herein, mayfurthermore comprise an adjuvant protein. In this context, an adjuvantprotein is preferably to be understood as any protein, which is capableto elicit an innate immune response as defined herein. Preferably, suchan innate immune response comprises an activation of a patternrecognition receptor, such as e.g. a receptor selected from theToll-like receptor (TLR) family, including e.g. a Toll like receptorselected from human TLR1 to TLR10 or from murine Toll like receptorsTLR1 to TLR13. Preferably, an innate immune response is elicited in amammal, more preferably in a human. Preferably, the adjuvant protein isselected from human adjuvant proteins or from pathogenic adjuvantproteins, in particular from bacterial adjuvant proteins. In addition,mRNA encoding human proteins involved in adjuvant effects may be used aswell.

Human adjuvant proteins, which may be encoded by the at least one RNA(molecule) of the complexed RNA as defined herein, typically compriseany human protein, which is capable of eliciting an innate immuneresponse (in a mammal), e.g. as a reaction of the binding of anexogenous TLR ligand to a TLR. More preferably, human adjuvant proteinswhich may be encoded by the complexed RNA of the present invention areselected from the group consisting of, without being limited thereto,cytokines which induce or enhance an innate immune response, includingIL-2, IL-12, IL-15, IL-18, IL-21CCL21, GM-CSF and TNF-alpha; cytokineswhich are released from macrophages, including IL-1, IL-6, IL-8, IL-12and TNF-alpha; from components of the complement system including C1 q,MBL, C1 r, C1s, C2b, Bb, D, MASP-1, MASP-2, C4b, C3b, C5a, C3a, C4a,C5b, C6, C7, C8, C9, CR1, CR2, CR3, CR4, C1qR, C1INH, C4bp, MCP, DAF, H,I, P and CD59; from proteins which are components of the signallingnetworks of the pattern recognition receptors including TLR and IL-1R1,whereas the components are ligands of the pattern recognition receptorsincluding IL-1alpha, IL-1 beta, Beta-defensin, heat shock proteins, suchas HSP10, HSP60, HSP65, HSP70, HSP75 and HSP90, gp96, Fibrinogen, TypIIIrepeat extra domain A of fibronectin; the receptors, including IL-1RI,TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11; thesignal transducers including components of the Small-GTPases signalling(RhoA, Ras, Rac1, Cdc42 etc.), components of the PIP signalling (PI3K,Src-Kinases, etc.), components of the MyD88-dependent signalling (MyD88,IRAK1, IRAK2, etc.), components of the MyD88-independent signalling(TICAM1, TICAM2 etc.); activated transcription factors including e.g.NF-κB, c-Fos, c-Jun, c-Myc; and induced target genes including e.g. IL-1alpha, IL-1 beta, Beta-Defensin, IL-6, IFN gamma, IFN alpha and IFNbeta; from costimulatory molecules, including CD28 or CD40-ligand orPD1; protein domains, including LAMP; cell surface proteins; or humanadjuvant proteins including CD80, CD81, CD86, trif, fit-3 ligand,thymopentin, Gp96 or fibronectin, etc., or any species homolog of any ofthe above human adjuvant proteins.

Pathogenic adjuvant proteins, which may be encoded by the at least oneRNA (molecule) of the complexed RNA as defined herein, typicallycomprise any pathogenic (adjuvant) protein, which is capable ofeliciting an innate immune response (in a mammal), more preferablyselected from pathogenic (adjuvant) proteins derived from bacteria,protozoa, viruses, or fungi, animals, etc., and even more preferablyfrom pathogenic adjuvant proteins selected from the group consisting of,without being limited thereto, bacterial proteins, protozoan proteins(e.g. profilin—like protein of Toxoplasma gondi), viral proteins, orfungal proteins, animal proteins, etc.

In this context, bacterial (adjuvant) proteins which may be encoded bythe at least one RNA (molecule) of the complexed RNA as defined herein,may comprise any bacterial protein, which is capable of eliciting aninnate immune response (preferably in a mammal). More preferably,bacterial (adjuvant) proteins, which may be encoded by the complexedRNA, may comprise bacterial adjuvant proteins selected from the groupconsisting of, without being limited thereto, bacterial flagellins,including flagellins from organisms including Agrobacterium, Aquifex,Azospirillum, Bacillus, Bartonella, Bordetella, Borrelia, Burkholderia,Campylobacter, Caulobacte, Clostridium, Escherichia, Helicobacter,Lachnospiraceae, Legionella, Listeria, Proteus, Pseudomonas, Rhizobium,Rhodobacter, Roseburia, Salmonella, Serpulina, Serratia, Shigella,Treponema, Vibrio, Wolinella, Yersinia, more preferably flagellins fromthe species, without being limited thereto, Agrobacterium tumefaciens,Aquifex pyrophilus, Azospinrllum brasilense, Bacillus subtilis, Bacillusthuringiensis, Bartonella baciliformis, Bordetella bronchiseptica,Borrelia burgdorfer Burkholderia cepacia, Campylobacter jejuni,Caulobacter crescentus, Clostridium botulinum strain Bennett clone 1,Escherichia coli Helicobacter pylori; Lachnospiraceae bacterium,Legionella pneumophila, Listeria monocytogenes, Proteus mirabilis,Pseudomonas aeroguinosa, Pseudomonas syringae, Rhizobium meliloti,Rhodobacter sphaeroides, Roseburia cecicola, Roseburis hominis,Salmonella typhimurium, Salmonella bongori Salmonella typhi, Salmonellaenteritidis, Serpulina hyodysenteriae, Serratia marcescens, Shigellaboydii, Treponema phagedenis, Vibrio alginolyticus, Vibrio cholerae,Vibrio parahaemolyticus, Wolinella succinogenes and Yersiniaenterocolitica.

Bacterial flagellins, which may be encoded by the at least one RNA(molecule) of the complexed RNA as defined herein, are particularlypreferred and may be selected from any bacterial flagellin showingadjuvant character, more preferably from bacterial flagellins selectedfrom the group consisting of bacterial heat shock proteins or chaperons,including Hsp60, Hsp70, Hsp90, Hsp100; OmpA (Outer membrane protein)from gram-negative bacteria; bacterial porins, including OmpF; bacterialtoxins, including pertussis toxin (PT) from Bordetella pertussis,pertussis adenylate cyclase toxin CyaA and CyaC from Bordetellapertussis, PT-9K/129G mutant from pertussis toxin, pertussis adenylatecyclase toxin CyaA and CyaC from Bordetella pertussis, tetanus toxin,cholera toxin (CT), cholera toxin B-subunit, CTK63 mutant from choleratoxin, CTE112K mutant from CT, Escherichia coli heat-labile enterotoxin(LT), B subunit from heat-labile enterotoxin (LTB) Escherichia coliheat-labile enterotoxin mutants with reduced toxicity, including LTK63,LTR72; phenol-soluble modulin; neutrophil-activating protein (HP-NAP)from Helicobacter pylori; Surfactant protein D; Outer surface protein Alipoprotein from Borrelia burgdorferi; Ag38 (38 kDa antigen) fromMycobacterium tuberculosis; proteins from bacterial fimbriae;Enterotoxin CT of Vibrio cholerae, Pilin from pili from gram negativebacteria, and Surfactant protein A; etc., or any species homolog of anyof the above bacterial (adjuvant) proteins.

Bacterial flagellins, which may be encoded by the at least one RNA(molecule) of the complexed RNA as defined herein, even more preferablycomprise a sequence selected from the group comprising any of thefollowing sequences as referred to their accession numbers:

organism species gene name accession No GI No AgrobacteriumAgrobacterium tumefaciens FlaD (flaD) U95165 GI:14278870 FlhB (flhB)FliG (fliG) FliN (fliN) FliM (fliM) MotA (motA) FlgF (flgF) FliI (fliI)FlgB (flgB) FlgC (flgC) FliE (fliE) FlgG (flgG) FlgA (flgA) FlgI (flgI)FlgH (flgH) FliL (fliL) FliP (fliP) FlaA (flaA) FlaB (flaB) FlaC (flaC)Aquifex Aquifex pyrophilus U17575 GI:596244 Azospirillum Azospirillumbrasilense Laf1 U26679 GI:1173509 Bacillus Bacillus subtilis hagAB033501 GI:14278870 Bacillus Bacillus thuringiensis flab X67138GI:46019718 Bartonella Bartonella bacilliformis L20677 GI:304184Bordetella Bordetella bronchiseptica flaA L13034 GI:289453 BorreliaBorrelia burgdorferi X16833 GI:39356 Burkholderia Burkholderia cepaciafliC AF011370 GI:2935154 Campylobacter Campylobacter jejuni flaA J05635GI:144197 flaB Caulobacter Caulobacter crescentus J01556 GI:144239Clostridium Clostridium botulinum FlaA DQ845000 GI:114054886 strainBennett clone 1 Escherichia Escherichia coli hag M14358 GI:146311 AJ884569 (EMBL-SVA) Helicobacter Helicobacter pylori flaA X60746 GI:43631Lachnospiraceae Lachnospiraceae bacterium DQ789131 GI:113911615Legionella Legionella pneumophila flaA X83232 GI:602877 ListeriaListeria monocytogenes flaA X65624 GI:44097 Proteus Proteus mirabilisFlaD (flaD) AF221596 GI:6959881 FlaA (flaA) FlaB (flaB) FliA (fliA) FliZ(fliZ) Pseudomonas Pseudomonas aeroguinosa flaA M57501 GI:151225Pseudomonas Pseudomonas syringae fliC EF544882 GI:146335619 RhizobiumRhizobium meliloti flaA M24526 GI:152220 flaB Rhodobacter Rhodobactersphaeroides fliC AF274346 GI:10716972 Roseburia Roseburia cecicolaM20983 GI:152535 Roseburia Roseburis hominis Fla2 DQ789141 GI:113911632Salmonella Salmonella typhimurium D13689 GI:217062 (NCBI ID) SalmonellaSalmonella bongori fliC AY603412 GI:51342390 Salmonella Salmonella typhiflag L21912 GI:397810 Salmonella Salmonella enteritidis fliC M84980GI:154015 Serpulina Serpulina hyodysenteriae flaB2 X63513 GI:450669Serratia Serratia marcescens hag M27219 GI:152826 Shigella Shigellaboydii fliC-SB D26165 GI:442485 Treponema Treponema phagedenis flaB2M94015 GI:155060 Vibrio Vibrio alginolyticus flaA EF125175 GI:119434395Vibrio s Vibrio parahaemolyticus AF069392 GI:7327274 Wolinella Wolinellasuccinogenes flag M82917 GI:155337 Yersinia Yersinia enterocoliticaL33467 GI:496295

Protozoan proteins, which may be encoded by the at least one RNA(molecule) of the complexed RNA as defined herein, may be selected fromany protozoan protein showing adjuvant character, more preferably, fromthe group consisting of, without being limited thereto, Tc52 fromTrypanosoma cruzi, PFTG from Trypanosoma gondii, Protozoan heat shockproteins, LeIF from Leishmania spp, profilin-like protein fromToxoplasma gondii, etc.

Viral proteins, which may be encoded by the at least one RNA (molecule)of the complexed RNA as defined herein, may be selected from any viralprotein showing adjuvant character, more preferably, from the groupconsisting of, without being limited thereto, Respiratory SyncytialVirus fusion glycoprotein (F-protein), envelope protein from MMT virus,mouse leukemia virus protein, Hemagglutinin protein of wild-type measlesvirus, etc.

Fungal proteins, which may be encoded by the at least one RNA (molecule)of the complexed RNA as defined herein, may be selected from any fungalprotein showing adjuvant character, more preferably, from the groupconsisting of, without being limited thereto, fungal immunomodulatoryprotein (FIP; LZ-8), etc.

Finally, pathogenic adjuvant proteins, which may be encoded by the atleast one RNA (molecule) of the complexed RNA as defined herein, mayfinally be selected from any further pathogenic protein showing adjuvantcharacter, more preferably, from the group consisting of, without beinglimited thereto, Keyhole limpet hemocyanin (KLH), OspA, etc.

The at least one RNA (molecule) of the complexed RNA of the presentinvention may alternatively encode an antigen. According to the presentinvention, the term “antigen” refers to a substance which is recognizedby the immune system and is capable of triggering an antigen-specificimmune response, e.g. by formation of antibodies. Antigens can beclassified according to their origin. Accordingly, there are two majorclasses of antigens: exogenous and endogenous antigens. Exogenousantigens are antigens that enter the cell or the body from outside (thecell or the body), for example by inhalation, ingestion or injection,etc. These antigens are internalized by antigen-presenting cells(“APCs”, such as dendritic cells or macrophages) and processed intofragments. APCs then present the fragments to T helper cells (e.g. CD4⁺)by the use of MHC II molecules on their surface. Recognition of theseantigen fragments by T cells leads to activation of the T cells andsecretion of cytokines. Cytokines are substances that can activateproliferation of immune cells such as cytotoxic T cells, B cells ormacrophages. In contrast, endogenous antigens are antigens which havebeen generated within the cell, e.g. as a result of normal cellmetabolism. Fragments of these antigens are presented on MHC I moleculeson the surface of APCs. These antigens are recognized by activatedantigen-specific cytotoxic CD8⁺ T cells. After recognition, those Tcells react in secretion of different toxins that cause lysis orapoptosis of the antigen-presenting cell. Endogenous antigens compriseantigens, e.g. proteins or peptides encoded by a foreign nucleic acidinside the cell as well as proteins or peptides encoded by the geneticinformation of the cell itself, or antigens from intracellularlyoccurring viruses. One class of endogenous antigens is the class oftumor antigens. Those antigens are presented by the MHC I molecules onthe surface of tumor cells. This class can be divided further intumor-specific antigens (TSAs) and tumor-associated-antigens (TAAs).TSAs can only be presented by tumor cells and never by normal “healthy”cells. They typically result from a tumor specific mutation. TAAs, whichare more common, are usually presented by both tumor and healthy cells.These antigens are recognized and the antigen-presenting cell can bedestroyed by cytotoxic T cells. Additionally, tumor antigens can alsooccur on the surface of the tumor in the form of e.g. a mutatedreceptor. In this case, they can be recognized by antibodies.

Antigens, which may be encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention, may include e.g. proteins,peptides or fragments thereof. Preferably, antigens are proteins andpeptides or fragments thereof, such as epitopes of those proteins orpeptides. Epitopes (also called “antigen determinants”), typically, arefragments located on the outer surface of such antigenic protein orpeptide structures having to 15, preferably 9 to 15, amino acids (B-cellepitopes and T-cell epitopes are typically presented on MHC molecules,wherein e.g. MHC-I typically presents epitopes with a length of about 9aa and MHC-II typically presents epitopes with a length of about 12-15aa). Furthermore, antigens encoded by the at least one RNA (molecule) ofthe complex according to the invention may also comprise any otherbiomolecule, e.g., lipids, carbohydrates, etc., which may be covalentlyor non-covalently attached to the RNA (molecule).

In accordance with the invention, antigens, which may be encoded by theat least one RNA (molecule) of the complexed RNA of the presentinvention, may be exogenous or endogenous antigens. Endogenous antigenscomprise antigens generated in the cell, especially in degenerate cellssuch as tumor cells. These antigens are referred to as “tumor antigens”.Preferably, without being restricted thereto, they are located on thesurface of the cell. Furthermore, “tumor antigens” means also antigensexpressed in cells which are (were) not by themselves (or originally notby themselves) degenerate but are associated with the supposed tumor.Antigens which are connected with tumor-supplying vessels or(re)formation thereof, in particular those antigens which are associatedwith neovascularization, e.g. growth factors, such as VEGF, bFGF etc.,are also included herein. Antigens connected with a tumor furthermoreinclude antigens from cells or tissues, typically embedding the tumor.Further, some substances (usually proteins or peptides) are expressed inpatients suffering (knowingly or not-knowingly) from a cancer diseaseand they occur in increased concentrations in the body fluids of saidpatients, e.g. proteins, which are associated with tumor cell invasionand migration. These substances are also referred to as “tumorantigens”, however they are not antigens in the stringent meaning of animmune response inducing substance. Use thereof is also encompassed bythe scope of the present invention.

Antigens, which may be encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention, may be exemplarily selected,without being restricted thereto, e.g. from any antigen suitable for thespecific purpose, e.g. from antigens, which are relevant (or causal) forspecific infection diseases, such as defined herein, from cancerantigens such as tumor specific surface antigens, from antigensexpressed in cancer diseases, from mutant antigens expressed in cancerdiseases, or from protein antigens involved in the etiology of furtherdiseases, e.g. autoimmune diseases, allergies, etc. E.g. these antigensmay be used to desensitize a patient by administering an antigen causingthe patient's allergic or autoimmune status.

Preferred exemplary antigenic (poly)peptides encoded by the at least oneRNA (molecule) of the complexed RNA as defined herein include all knownantigenic peptides, for example tumour antigens, etc. Specific examplesof tumour antigens are inter alia tumour-specific surface antigens(TSSAs), for example 5T4, alpha5beta1-integrin, 707-AP, AFP, ART-4,B7H4, BAGE, Bcr-abl, MN/C IX antigen, CA125, CAMEL, CAP-1, CASP-8,beta-catenin/m, CD4, CD19, CD20, CD22, CD25, CDC27/m, CD 30, CD33, CD52,CD56, CD80, CDK4/m, CEA, CT, Cyp-B, DAM, EGFR, ErbB3, ELF2M, EMMPRIN,EpCam, ETV6-AML1, G250, GAGE, GnT-V, Gp100, HAGE, HER-2/new,HLA-A*0201-R1701, HPV-E7, HSP70-2M, HAST-2, hTERT (or hTRT), iCE,IGF-1R, IL-2R, IL-5, KIAA0205, LAGE, LDLR/FUT, MAGE, MART-1/melan-A,MART-2/Ski, MC1R, myosin/m, MUC1, MUM-1, -2, -3, NA88-A, PAP,proteinase-3, p190 minor bcr-abl, Pml/RARalpha, PRAME, PSA, PSM, PSMA,RAGE, RU1 or RU2, SAGE, SART-1 or SART-3, survivin, TEUAML1, TGFbeta,TPVm, TRP-1, TRP-2, TRP-2/INT2, VEGF and WT1, or from sequences such as,for example, NY-Eso-1 or NY-Eso-B. Any class of tumor antigens issuitable for the purpose of the present invention, e.g. tumor antigensknown to be involved in neovascularization, influencing theextracellular matrix structure etc. Fragments and analogues of the aboveantigens are also encompassed.

Examples of tumor antigens which may be encoded by the at least one RNA(molecule) of the complexed RNA of the present invention are shown inTables 1 and 2 below. These tables illustrate specific (protein)antigens (i.e. “tumor antigens”) with respect to the cancer disease,they are associated with. According to the invention, the terms “cancerdiseases” and “tumor diseases” are used synonymously herein.

TABLE 1 Antigens expressed in cancer diseases Tumor antigen NameExpression site 5T4 colorectal cancer, gastric cancer, ovarian cancer707-AP 707 alanine proline melanoma 9D7 renal cell carcinoma AFPalpha-fetoprotein hepatocellular carcinoma, gallbladder cancer,testicular cancer, ovarian cancer, bladder cancer AlbZIP HPG1 prostatecancer alpha5beta1- Integrin alpha5beta6- colon cancer Integrinalpha-methylacyl- prostate cancer coenzyme A racemase ART-4adenocarcinoma antigen lung cancer, head and neck cancer, recognized byT cells 4 leukemia, esophageal cancer, gastric cancer, cervical cancer,ovarian cancer, breast cancer, squamous cell carcinoma B7H4 ovariancancer BAGE-1 B antigen bladder cancer, head and neck cancer, lungcancer, melanoma, squamous cell carcinoma BCL-2 leukemia BING-4 melanomaCA 15-3/CA 27-29 breast cancer, ovary cancer, lung cancer, prostatecancer CA 19-9 gastric cancer, pancreatic cancer, liver cancer, breastcancer, gallbladder cancer, colon cancer, ovary cancer, lung cancer CA72-4 ovarian cancer CA125 ovarian cancer, colorectal cancer, gastriccancer, liver cancer, pancreatic cancer, uterus cancer, cervixcarcinoma, colon cancer, breast cancer, lung cancer calreticulin bladdercancer CAMEL CTL-recognized antigen on melanoma melanoma CASP-8caspase-8 head and neck cancer cathepsin B breast cancer cathepsin Lbreast cancer CD19 B-cell malignancies CD20 CD22 CD25 CD30 CD33 CD4 CD52CD55 CD56 CD80 CEA carcinoembryonic antigen gut carcinoma, colorectalcancer, colon cancer, hepatocellular cancer, lung cancer, breast cancer,thyroid cancer, pancreatic cancer, liver cancer cervix cancer, bladdercancer, melanoma CLCA2 calcium-activated chloride lung cancer channel-2CML28 leukemia Coactosin-like pancreatic cancer protein Collagen XXIIIprostate cancer COX-2 ovarian cancer, breast cancer, colorectal cancerCT-9/BRD6 bromodomain testis-specific protein Cten C-terminaltensin-like protein prostate cancer cyclin B1 cyclin D1 ovarian cancercyp-B cyclophilin B bladder cancer, lung cancer, T-cell leukemia,squamous cell carcinoma, CYPB1 cytochrom P450 1B1 leukemia DAM-10/MAGE-differentiation antigen melanoma melanoma, skin tumors, ovarian cancer,B1 10 lung cancer DAM-6/MAGE-B2 differentiation antigen melanomamelanoma, skin tumors, ovarian cancer, 6 lung cancer EGFR/Her1 lungcancer, ovarian cancer, head and neck cancer, colon cancer, pancreaticcancer, breast cancer EMMPRIN tumor cell-associated lung cancer, breastcancer, bladder extracellular matrix cancer, ovarian cancer, braincancer, metalloproteinase inducer/ lymphoma EpCam epithelial celladhesion molecule ovarian cancer, breast cancer, colon cancer, lungcancer EphA2 ephrin type-A receptor 2 glioma EphA3 ephrin type-Areceptor 2 melanoma, sarcoma, lung cancer ErbB3 breast cancer EZH2(enhancer of Zeste homolog 2) endometrium cancer, melanoma, prostatecancer, breast cancer FGF-5 fibroblast growth factor-5 renal cellcarcinoma, breast cancer, prostate cancer FN fibronectin melanoma Fra-1Fos-related antigen-1 breast cancer, esophageal cancer, renal cellcarcinoma, thyroid cancer G250/CAIX glycoprotein 250 leukemia, renalcell carcinoma, head and neck cancer, colon cancer, ovarian cancer,cervical cancer GAGE-1 G antigen 1 bladder cancer, lung cancer, sarcoma,melanoma, head and neck cancer GAGE-2 G antigen 2 bladder cancer, lungcancer, sarcoma, melanoma, head and neck cancer GAGE-3 G antigen 3bladder cancer, lung cancer, sarcoma, melanoma, head and neck cancerGAGE-4 G antigen 4 bladder cancer, lung cancer, sarcoma, melanoma, headand neck cancer GAGE-5 G antigen 5 bladder cancer, lung cancer, sarcoma,melanoma, head and neck cancer GAGE-6 G antigen 6 bladder cancer, lungcancer, sarcoma, melanoma, head and neck cancer GAGE-7b G antigen 7bbladder cancer, lung cancer, sarcoma, melanoma, head and neck cancerGAGE-8 G antigen 8 bladder cancer, lung cancer, sarcoma, melanoma, headand neck cancer GDEP gene differentially expressed in prostate cancerprostate GnT-V N-acetylglucosaminyltransferase glioma, melanoma V gp100glycoprotein 100 kDa melanoma GPC3 glypican 3 hepatocellular carcinoma,melanoma HAGE helicase antigen bladder cancer HAST-2 human signet ringtumor-2 hepsin prostate Her2/neu/ErbB2 human epidermal receptor- breastcancer, bladder cancer, 2/neurological melanoma, ovarian cancer,pancreas cancer, gastric cancer HERV-K-MEL melanoma HNE human neutrophilelastase leukemia homeobox NKX prostate cancer 3.1 HOM-TES- ovariancancer 14/SCP-1 HOM-TES-85 HPV-E6 cervical cancer HPV-E7 cervical cancerHST-2 gastric cancer hTERT human telomerase reverse breast cancer,melanoma, lung cancer, transcriptase ovarian cancer, sarcoma,Non-Hodgkin- lymphoma, acute leukemia iCE intestinal carboxyl esteraserenal cell carcinoma IGF-1R colorectal cancer IL-13Ra2 interleukin 13receptor alpha 2 glioblastoma chain IL-2R colorectal cancer IL-5immature laminin renal cell carcinoma receptor kallikrein 2 prostatecancer kallikrein 4 prostate cancer Ki67 prostate cancer, breast cancer,Non- Hodgkin-lymphoma, melanoma KIAA0205 bladder cancer KK-LC-1Kita-kyushu lung cancer antigen lung cancer 1 KM-HN-1 tongue cancer,hepatocellular carcinomas, melanoma, gastric cancer, esophageal, coloncancer, pancreatic cancer LAGE-1 L antigen bladder cancer, head and neckcancer, melanoma livin bladder cancer, melanoma MAGE-A1 melanomaantigen-A1 bladder cancer, head and neck cancer, melanoma, colon cancer,lung cancer, sarcoma, leukemia MAGE-A10 melanoma antigen-A10 bladdercancer, head and neck cancer, melanoma, colon cancer, lung cancer,sarcoma, leukemia MAGE-A12 melanoma antigen-A12 bladder cancer, head andneck cancer, melanoma, colon cancer, lung cancer, sarcoma, leukemia,prostate cancer, myeloma, brain tumors MAGE-A2 melanoma antigen-A2bladder cancer, head and neck cancer, melanoma, colon cancer, lungcancer, sarcoma, leukemia MAGE-A3 melanoma antigen-A3 bladder cancer,head and neck cancer, melanoma, colon cancer, lung cancer, sarcoma,leukemia MAGE-A4 melanoma antigen-A4 bladder cancer, head and neckcancer, melanoma, colon cancer, lung cancer, sarcoma, leukemia MAGE-A6melanoma antigen-A6 bladder cancer, head and neck cancer, melanoma,colon cancer, lung cancer, sarcoma, leukemia MAGE-A9 melanoma-antigen-A9bladder cancer, head and neck cancer, melanoma, colon cancer, lungcancer, sarcoma, leukemia MAGE-B1 melanoma-antigen-B1 melanoma MAGE-B10melanoma-antigen-B10 melanoma MAGE-B16 melanoma-antigen-B16 melanomaMAGE-B17 melanoma-antigen-B17 melanoma MAGE-B2 melanoma-antigen-B2melanoma MAGE-B3 melanoma-antigen-B3 melanoma MAGE-B4melanoma-antigen-B4 melanoma MAGE-B5 melanoma-antigen-B5 melanomaMAGE-B6 melanoma-antigen-B6 melanoma MAGE-C1 melanoma-antigen-C1 bladdercancer, melanoma MAGE-C2 melanoma-antigen-C2 melanoma MAGE-C3melanoma-antigen-C3 melanoma MAGE-D1 melanoma-antigen-D1 melanomaMAGE-D2 melanoma-antigen-D2 melanoma MAGE-D4 melanoma-antigen-D4melanoma MAGE-E1 melanoma-antigen-E1 bladder cancer, melanoma MAGE-E2melanoma-antigen-E2 melanoma MAGE-F1 melanoma-antigen-F1 melanomaMAGE-H1 melanoma-antigen-H1 melanoma MAGEL2 MAGE-like 2 melanomamammaglobin A breast cancer MART-1/Melan-A melanoma antigen recognizedby melanoma T cells-1/melanoma antigen A MART-2 melanoma antigenrecognized by melanoma T cells-2 matrix protein 22 bladder cancer MC1Rmelanocortin 1 receptor melanoma M-CSF macrophage colony-stimulatingovarian cancer factor gene mesothelin ovarian cancer MG50/PXDN breastcancer, glioblastoma, melanoma MMP 11 M-phase phosphoprotein 11 leukemiaMN/CA IX-antigen renal cell carcinoma MRP-3 multidrugresistance-associated lung cancer protein 3 MUC1 mucin 1 breast cancerMUC2 mucin 2 breast cancer, ovarian cancer, pancreatic cancer NA88-A NAcDNA clone of patient M88 melanoma N- acetylglucosaminyl- transferase-VNeo-PAP Neo-poly(A) polymerase NGEP prostate cancer NMP22 bladder cancerNPM/ALK nucleophosmin/anaplastic lymphoma kinase fusion protein NSEneuron-specific enolase small cell cancer of lung, neuroblastoma, Wilm'tumor, melanoma, thyroid cancer, kidney cancer, testicle cancer,pancreas cancer NY-ESO-1 New York esophageous 1 bladder cancer, head andneck cancer, melanoma, sarcoma, B-lymphoma, hepatoma, pancreatic cancer,ovarian cancer, breast cancer NY-ESO-B OA1 ocular albinism type 1protein melanoma OFA-iLRP oncofetal antigen-immature leukemia lamininreceptor OGT O-linked N-acetylglucosamine transferase gene OS-9osteocalcin prostate cancer osteopontin prostate cancer, breast cancer,ovarian cancer p15 protein 15 p15 melanoma p190 minor bcr- abl p53PAGE-4 prostate GAGE-like protein-4 prostate cancer PAI-1 plasminogenacitvator inhibitor 1 breast cancer PAI-2 plasminogen acitvatorinhibitor 2 breast cancer PAP prostate acic phosphatase prostate cancerPART-1 prostate cancer PATE prostate cancer PDEF prostate cancerPim-1-Kinase Pin1 Propyl isomerase prostate cancer POTE prostate cancerPRAME preferentially expressed antigen melanoma, lung cancer, leukemia,head of melanoma and neck cancer, renal cell carcinoma, sarcoma prosteinprostate cancer proteinase-3 PSA prostate-specific antigen prostatecancer PSCA prostate cancer PSGR prostate cancer PSM PSMAprostate-specific membrane prostate cancer antigen RAGE-1 renal antigenbladder cancer, renal cancer, sarcoma, colon cancer RHAMM/CD168 receptorfor hyaluronic acid leukemia mediated motility RU1 renal ubiquitous 1bladder cancer, melanoma, renal cancer RU2 renal ubiquitous 1 bladdercancer, melanoma, sarcoma, brain tumour, esophagel cancer, renal cancer,colon cancer, breast cancer S-100 melanoma SAGE sarcoma antigen SART-1squamous antigen rejecting tumor esophageal cancer, head and neck 1cancer, lung cancer, uterine cancer SART-2 squamous antigen rejectingtumor head and neck cancer, lung cancer, renal 1 cell carcinoma,melanoma, brain tumour SART-3 squamous antigen rejecting tumor head andneck cancer, lung cancer, 1 leukemia, melanoma, esophageal cancer SCCsquamous cell carcinoma antigen lung cancer Sp17 sperm protein 17multiple myeloma SSX-1 synovial sarcoma X breakpoint 1 hepatocellularcell carcinom, breast cancer SSX-2/HOM-MEL- synovial sarcoma Xbreakpoint 2 breast cancer 40 SSX-4 synovial sarcoma X breakpoint 4bladder cancer, hepatocellular cell carcinoma, breast cancer STAMP-1prostate cancer STEAP six transmembrane epithelial prostate cancerantigen prostate survivin bladder cancer survivin-2B intron 2-retainingsurvivin bladder cancer TA-90 melanoma TAG-72 prostate carcinoma TARPprostate cancer TGFb TGFbeta TGFbRII TGFbeta receptor II TGM-4prostate-specific transglutaminase prostate cancer TRAG-3 taxolresistant associated protein breast cancer, leukemia, and melanoma 3 TRGtestin-related gene TRP-1 tyrosine related protein 1 melanoma TRP-2/6bTRP-2/novel exon 6b melanoma, glioblastoma TRP-2/INT2 TRP-2/intron 2melanoma, glioblastoma Trp-p8 prostate cancer Tyrosinase melanoma UPAurokinase-type plasminogen breast cancer activator VEGF vascularendothelial growth factor VEGFR-2/FLK-1 vascular endothelial growthfactor receptor-2 WT1 Wilm' tumor gene gastric cancer, colon cancer,lung cancer, breast cancer, ovarian cancer, leukemia

TABLE 2 Mutant antigens expressed in cancer diseases Mutant antigen NameExpression site alpha-actinin-4/m lung carcinoma ARTC1/m melanomabcr/abl breakpoint cluster region- CML Abelson fusion proteinbeta-Catenin/m beta-Catenin melanoma BRCA1/m breast cancer BRCA2/mbreast cancer CASP-5/m colorectal cancer, gastric cancer, endometrialcarcinoma CASP-8/m head and neck cancer, squamous cell carcinoma CDC27/mcell-divison-cycle 27 CDK4/m cyclin-dependent kinase 4 melanoma CDKN2A/mmelanoma CML66 CML COA-1/m colorectal cancer DEK-CAN fusion protein AMLEFTUD2/m melanoma ELF2/m Elongation factor 2 lung squamous cellcarcinoma ETV6-AML1 Ets variant gene6/acute myeloid ALL leukemia 1 genefusion protein FN1/m fibronectin 1 melanoma GPNMB/m melanoma HLA-A*0201-arginine to isoleucine exchange renal cell carcinoma R170I at residue170 of the alpha-helix of the alpha2-domain in the HLA-A2 gene HLA-A11/mmelanoma HLA-A2/m renal cell carcinoma HSP70-2M heat shock protein 70-2mutated renal cell carcinoma, melanoma, neuroblastoma KIAA0205/m bladdertumor K-Ras/m pancreatic carcinoma, colorectal carcinoma LDLR-FUTLDR-Fucosyltransferase fusion melanoma protein MART2/m melanoma ME1/mnon-small cell lung carcinoma MUM-1/m melanoma ubiquitous mutated 1melanoma MUM-2/m melanoma ubiquitous mutated 2 melanoma MUM-3/m melanomaubiquitous mutated 3 melanoma Myosin class I/m melanoma neo-PAP/mmelanoma NFYC/m lung squamous cell carcinoma N-Ras/m melanoma OGT/mcolorectal carcinoma OS-9/m melanoma p53/m Pml/RARa promyelocyticleukemia/retinoic APL, PML acid receptor alpha PRDX5/m melanoma PTPRK/mreceptor-type protein-tyrosine melanoma phosphatase kappa RBAF600/mmelanoma SIRT2/m melanoma SYT-SSX-1 synaptotagmin I/synovial sarcomasarcoma X fusion protein SYT-SSX-2 synaptotagmin I/synovial sarcomasarcoma X fusion protein TEL-AML1 translocation Ets-family AMLleukemia/acute myeloid leukemia 1 fusion protein TGFbRII TGFbetareceptor II colorectal carcinoma TPI/m triosephosphate isomerasemelanoma

In a preferred embodiment according to the invention, examples of tumorantigens which may be encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention are selected from the groupconsisting of 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, 5 1-integrin, 56-integrin, -actinin-4/m, methylacyl-coenzyme A racemase, ART-4,ARTC1/m, B7H4, BAGE-1, BCL-2, bcr/abl, catenin/m, BING-4, BRCA1/m,BRCA2/m, CA 15-3/CA 27-29, CA 19-9, CA72-4, CA125, calreticulin, CAMEL,CASP-8/m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33,CD4, CD52, CD55, CD56, CD80, CDC27/m, CDK4/m, CDKN2A/m, CEA, CLCA2,CML28, CML66, COA-1/m, coactosin-like protein, collage XXIII, COX-2,CT-9/BRD6, Cten, cyclin B1, cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6,DEK-CAN, EFTUD2/m, EGFR, ELF2/m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3,ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3,GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3,GPNMB/m, HAGE, HAST-2, hepsin, Her2/neu, HERV-K-MEL, HLA-A*0201-R171,HLA-A1/m, HLA-A2/m, HNE, homeobox NKX3.1, HOM-TES-14/SCP-1, HOM-TES-85,HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Rα2, IL-2R,IL-5, immature laminin receptor, kallikrein-2, kallikrein-4, Ki67,KIAA0205, KIAA0205/m, KK-LC-1, K-Ras/m, LAGE-A1, LDLR-FUT, MAGE-A1,MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12,MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10,MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2,MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, mammaglobin A,MART-1/melan-A, MART-2, MART-2/m, matrix protein 22, MC1R, M-CSF, ME1/m,mesothelin, MG50/PXDN, MMP11, MN/CA IX-antigen, MRP-3, MUC-1, MUC-2,MUM-1/m, MUM-2/m, MUM-3/m, myosin class I/m, NA88-A,N-acetylglucosaminyltransferase-V, Neo-PAP, Neo-PAP/m, NFYC/m, NGEP,NMP22, NPM/ALK, N-Ras/m, NSE, NY-ESO-1, NY-ESO-B, OA1, OFA-iLRP, OGT,OGT/m, OS-9, OS-9/m, osteocalcin, osteopontin, p15, p190 minor bcr-abl,p53, p53/m, PAGE-4, PAI-1, PAI-2, PART-1, PATE, PDEF, Pim-1-Kinase,Pin-1, Pml/PARα, POTE, PRAME, PRDX5/m, prostein, proteinase-3, PSA,PSCA, PSGR, PSM, PSMA, PTPRK/m, RAGE-1, RBAF600/m, RHAMM/CD168, RU1,RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2/m, Sp17, SSX-1,SSX-2/HOM-MEL-40, SSX-4, STAMP-1, STEAP, survivin, survivin-2B,SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGFβ, TGFβRII,TGM-4, TPI/m, TRAG-3, TRG, TRP-1, TRP-2/6b, TRP/INT2, TRP-p8,tyrosinase, UPA, VEGF, VEGFR-2/FLK-1, and WT1.

In a preferred embodiment according to the invention, examples of tumorantigens which may be encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention are selected from the groupconsisting of MAGE-A1 [accession number M77481], MAGE-A6 [accessionnumber NM_005363], melan-A [accession number NM_005511], GP100[accession number M77348], tyrosinase [accession number NM_000372],survivin [accession number AF077350], CEA [accession number NM_004363],Her-2/neu [accession number M11730], WT1 [accession number NM_000378],PRAME [accession number NM_006115], EGFRI (epidermal growth factorreceptor 1) [accession number AF288738], mucin-1 [accession numberNM_002456] and SEC61G [accession number NM_014302].

As a further alternative, the at least one RNA (molecule) of thecomplexed RNA of the present invention may encode an antibody. Accordingto the present invention, such an antibody may be selected from anyantibody, e.g. any recombinantly produced or naturally occurringantibodies, known in the art, in particular antibodies suitable fortherapeutic, diagnostic or scientific purposes, or antibodies which havebeen identified in relation to specific cancer diseases. Herein, theterm “antibody” is used in its broadest sense and specifically coversmonoclonal and polyclonal antibodies (including agonist, antagonist, andblocking or neutralizing antibodies) and antibody species withpolyepitopic specificity. According to the invention, “antibody”typically comprises any antibody known in the art (e.g. IgM, IgD, IgG,IgA and IgE antibodies), such as naturally occurring antibodies,antibodies generated by immunization in a host organism, antibodieswhich were isolated and identified from naturally occurring antibodiesor antibodies generated by immunization in a host organism andrecombinantly produced by biomolecular methods known in the art, as wellas chimeric antibodies, human antibodies, humanized antibodies,bispecific antibodies, intrabodies, i.e. antibodies expressed in cellsand optionally localized in specific cell compartments, and fragmentsand variants of the aforementioned antibodies. In general, an antibodyconsists of a light chain and a heavy chain both having variable andconstant domains. The light chain consists of an N-terminal variabledomain, V_(L), and a C-terminal constant domain, C_(L). In contrast, theheavy chain of the IgG antibody, for example, is comprised of anN-terminal variable domain, V_(H), and three constant domains, C_(H)1,C_(H)2 und C_(H)3. Single chain antibodies may be encoded by the atleast one RNA (molecule) of the complexed RNA as defined herein as well,preferably by a single-stranded RNA, more preferably by an mRNA.

According to a first alternative, the at least one RNA (molecule) of thecomplexed RNA of the present invention may encode a polyclonal antibody.In this context, the term, “polyclonal antibody” typically meansmixtures of antibodies directed to specific antigens or immunogens orepitopes of a protein which were generated by immunization of a hostorganism, such as a mammal, e.g. including goat, cattle, swine, dog,cat, donkey, monkey, ape, a rodent such as a mouse, hamster and rabbit.Polyclonal antibodies are generally not identical, and thus usuallyrecognize different epitopes or regions from the same antigen. Thus, insuch a case, typically a mixture (a composition) of different RNAmolecules complexed as claimed by the present invention will be applied,each encoding a specific (monoclonal) antibody being directed tospecific antigens or immunogens or epitopes of a protein.

According to a further alternative, the at least one RNA (molecule) ofthe complexed RNA of the present invention may encode a monoclonalantibody. The term “monoclonal antibody” herein typically refers to anantibody obtained from a population of substantially homogeneousantibodies, i.e., the individual antibodies comprising the populationare identical except for possible naturally-occurring mutations that maybe present in minor amounts. Monoclonal antibodies are highly specific,being directed to a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which typically includedifferent antibodies directed to different determinants (epitopes), eachmonoclonal antibody is directed to a single determinant on the antigen.For example, monoclonal antibodies as defined above may be made by thehybridoma method first described by Kohler and Milstein, Nature, 256:495(1975), or may be made by recombinant DNA methods, e.g. as described inU.S. Pat. No. 4,816,567. “Monoclonal antibodies” may also be isolatedfrom phage libraries generated using the techniques described inMcCafferty et al., Nature, 348:552-554 (1990), for example. According toKohler and Milstein, an immunogen (antigen) of interest is injected intoa host such as a mouse and B-cell lymphocytes produced in response tothe immunogen are harvested after a period of time. The B-cells arecombined with myeloma cells obtained from mouse and introduced into amedium which permits the B-cells to fuse with the myeloma cells,producing hybridomas. These fused cells (hybridomas) are then placed inseparate wells in microtiter plates and grown to produce monoclonalantibodies. The monoclonal antibodies are tested to determine which ofthem are suitable for detecting the antigen of interest. After beingselected, the monoclonal antibodies can be grown in cell cultures or byinjecting the hybridomas into mice. However, for the purposes of thepresent invention, the peptide sequences of these monoclonal antibodieshave to be sequenced and RNA sequences encoding these antibodies may beprepared according to procedures well known in the art.

For therapeutical purposes in humans, non-human monoclonal or polyclonalantibodies, such as murine antibodies may also be encoded by the atleast one RNA (molecule) of the complexed RNA of the present invention.However, such antibodies are typically only of limited use, since theygenerally induce an immune response by production of human antibodiesdirected to the said non-human antibodies, in the human body. Therefore,a particular non-human antibody can only be administered once to thehuman. To solve this problem, chimeric, humanized non-human and humanantibodies can be encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention. “Chimeric” antibodies, which maybe encoded by the at least one RNA (molecule) of the complexed RNA ofthe present invention, are preferably antibodies in which the constantdomains of an antibody described above are replaced by sequences ofantibodies from other organisms, preferably human sequences. “Humanized”(non-human) antibodies, which may be also encoded by the at least oneRNA (molecule) of the complexed RNA of the present invention, areantibodies in which the constant and variable domains (except for thehypervariable domains) described above of an antibody are replaced byhuman sequences. According to another alternative, the at least one RNA(molecule) of the complexed RNA of the present invention may encodehuman antibodies, i.e. antibodies having only human sequences. Suchhuman antibodies can be isolated from human tissues or from immunizednon-human host organisms which are transgene for the human IgG genelocus, sequenced RNA sequences may be prepared according to procedureswell known in the art. Additionally, human antibodies can be provided bythe use of a phage display.

In addition, the at least one RNA (molecule) of the complexed RNA of thepresent invention may encode bispecific antibodies. “Bispecific”antibodies in context of the invention are preferably antibodies whichact as an adaptor between an effector and a respective target, e.g. forthe purposes of recruiting effector molecules such as toxins, drugs,cytokines etc., targeting effector cells such as CTL, NK cells,makrophages, granulocytes, etc. (see for review: Kontermann R. E., ActaPharmacol. Sin, 2005, 26(1): 1-9). Bispecific antibodies as describedherein are, in general, configured to recognize, e.g. two differentantigens, immunogens, epitopes, drugs, cells (or receptors on cells), orother molecules (or structures) as described above. Bispecificity meansherewith that the antigen-binding regions of the antibodies are specificfor two different epitopes. Thus, different antigens, immunogens orepitopes, etc. can be brought close together, what, optionally, allows adirect interaction of the two components. For example, different cellssuch as effector cells and target cells can be connected via abispecific antibody. Encompassed, but not limited, by the presentinvention are antibodies or fragments thereof which bind, on the onehand, a soluble antigen as described herein, and, on the other hand, anantigen or receptor on the surface of a tumor cell.

In summary, according to the invention, the at least one RNA (molecule)of the complexed RNA of the present invention may also encode antibodiesas defined above. Since these antibodies are intracellularly expressedantibodies, i.e. antibodies which are encoded by nucleic acids localizedin specific compartments of the cell and also expressed there, suchantibodies may also be termed intrabodies.

Antibodies as encoded by the at least one RNA (molecule) of thecomplexed RNA of the present invention may preferably comprisefull-length antibodies, i.e. antibodies composed of the full heavy andfull light chains, as described above. However, derivatives ofantibodies such as antibody fragments, variants or adducts may beencoded by the above at least one RNA of the complexed RNA in accordancewith the present invention.

The at least one RNA (molecule) of the complexed RNA of the presentinvention may also encode antibody fragments selected from Fab, Fab′,F(ab′)₂, Fc, Facb, pFc′, Fd and Fv fragments of the aforementionedantibodies. In general, antibody fragments are known in the art. Forexample, a Fab (“fragment, antigen binding”) fragment is composed of oneconstant and one variable domain of each of the heavy and the lightchain. The two variable domains bind the epitope on specific antigens.The two chains are connected via a disulfide linkage. A scFv (“singlechain variable fragment”) fragment, for example, typically consists ofthe variable domains of the light and heavy chains. The domains arelinked by an artificial linkage, in general a polypeptide linkage suchas a peptide composed of 15-25 glycine, proline and/or serine residues.

According to the invention, the at least one RNA (molecule) of thecomplexed RNA of the present invention may encode fragments and/orvariants of the aforementioned therapeutically active proteins, antigensor antibodies, wherein the fragments and/or variants may have a sequenceidentity to one of the aforementioned therapeutically active proteins,antigens or antibodies of at least 70%, 80% or 85%, preferably at least90%, more preferably at least 95% and most preferably at least 99% overthe whole length of the coding nucleic acid or amino acid sequencesencoding these therapeutically active proteins, antigens or antibodies.Preferably, the fragments and/or variants have the same biologicalfunction or specific activity compared to the full-length nativetherapeutically active proteins, antigens or antibodies, e.g. specificbinding capacity (e.g. of particular antigens), catalytic activity (e.g.of therapeutically active proteins), etc. In this context, “biologicalfunction” of antibodies described herein also comprises neutralizationof antigens, complement activation or opsonization. Thereby, antibodiestypically recognize either native epitopes on the cell surface or freeantigens. Antibodies as defined above can interact with thecell-presenting antigens and initiate different defense mechanisms. Onthe one hand, the antibody can initiate signaling mechanisms in thetargeted cell that leads to the cell's self-destruction (apoptosis). Onthe other hand, it can mark the cell in such a way that other componentsor effector cells of the body's immune system can recognize and attack.The attack mechanisms are referred to as antibody-dependentcomplement-mediated cytotoxicity (CMC) and antibody-dependent cellularcytotoxicity (ADCC). ADCC involves a recognition of the antibody byimmune cells that engage the antibody-marked cells and either throughtheir direct action, or through the recruitment of other cell types,lead to the tagged-cell's death. CMC is a process where a cascade ofdifferent complement proteins becomes activated, usually when severalantibodies are in close proximity to each other, either resulting incell lysis or attracting other immune cells to this location foreffector cell function. In the neutralization of an antigen, theantibody can bind an antigen and neutralize the same. Suchneutralization reaction, in turn, leads in general to blocking of theantibody. Thus, the antibody can bind only one antigen, or, in case of abispecific antibody, two antigens. In particular, scFv antibodyfragments are useful for neutralization reactions because they don'tcontain the functionalities of the constant domain of an antibody. Inthe complement activation, the complex system of complement proteins canbe activated via binding of an antibody which is independent of the Fcpart of an antibody. End products of the complement cascade result inlysis of the cell and generation of an inflammatory milieu. In theopsonization, pathogens or other non-cellular particles are madeaccessible to phagocytes via binding the constant domain of an antibody.Alternatively, cells recognized as foreign can be lysed viaantibody-dependent cell-mediated cytotoxicity (ADCC). In particular,NK-cells can display lysis functions by activating Fc receptors.

In order to determine the percentage to which two RNA sequences (nucleicor amino acid) are identical, the sequences can be aligned in order tobe subsequently compared to one another. Therefore, e.g. gaps can beinserted into the sequence of the first sequence and the component atthe corresponding position of the second sequence can be compared. If aposition in the first sequence is occupied by the same component as isthe case at a position in the second sequence, the two sequences areidentical at this position. The percentage to which two sequences areidentical is a function of the number of identical positions divided bythe total number of positions.

The percentage to which two sequences are identical can be determinedusing a mathematical algorithm. A preferred, but not limiting, exampleof a mathematical algorithm which can be used is the algorithm of Karlinet al. (1993), PNAS USA, 90:5873-5877 or Altschul et al. (1997), NucleicAcids Res, 25:3389-3402. Such an algorithm is integrated in the BLASTprogram. Sequences which are identical to the sequences of the RNA ofthe complexed RNA of the present invention to a certain extent can beidentified by this program.

Those at least one RNA molecules (of the complexed RNA of the presentinvention) encoding amino acid sequences which have (a) conservativesubstitution(s) compared to the physiological sequence in particularfall under the term variants. Substitutions in which encoded amino acidswhich originate from the same class are exchanged for one another arecalled conservative substitutions. In particular, these are encodedamino acids encoded aliphatic side chains, positively or negativelycharged side chains, aromatic groups in the side chains or encoded aminoacids, the side chains of which can enter into hydrogen bridges, e.g.side chains which have a hydroxyl function. This means that e.g. anamino acid having a polar side chain is replaced by another amino acidhaving a likewise polar side chain, or, for example, an amino acidcharacterized by a hydrophobic side chain is substituted by anotheramino acid having a likewise hydrophobic side chain (e.g. serine(threonine) by threonine (serine) or leucine (isoleucine) by isoleucine(leucine)). Insertions and substitutions are possible, in particular, atthose sequence positions which cause no modification to thethree-dimensional structure or do not affect the binding region.Modifications to a three-dimensional structure by insertion(s) ordeletion(s) can easily be determined e.g. using CD spectra (circulardichroism spectra) (Urry, 1985, Absorption, Circular Dichroism and ORDof Polypeptides, in: Modern Physical Methods in Biochemistry, Neubergeret a. (ed.), Elsevier, Amsterdam).

Immunostimulatory RNA

According to a third embodiment, the at least one RNA (molecule) of thecomplexed RNA of the present invention may be an immunostimulatory RNA.Thereby, the immunostimulatory RNA may exhibit an immunostimulatoryeffect already prior to complexation of the RNA with the inventiveoligopeptide according to formula (I) as defined above, or, morepreferably, an immunostimulatory effect of the RNA as used herein can beenhanced or even induced by complexation of the RNA with the inventiveoligopeptide according to formula (I) as defined above. Theimmunostimulatory RNA of the complexed RNA of the present invention maybe any RNA, e.g. a coding RNA, as defined above. Preferably, theimmunostimulatory RNA may be a single-stranded, a double-stranded or apartially double-stranded RNA, more preferably a single-stranded RNA,and/or a circular or linear RNA, more preferably a linear RNA. Morepreferably, the immunostimulatory RNA may be a (linear) single-strandedRNA. Even more preferably, the immunostimulatory RNA may be a ((linear)single-stranded) messenger RNA (mRNA). An immunostimulatory RNA may alsooccur as a short RNA oligonucleotide as defined above.

An immunostimulatory RNA as used herein may furthermore be selected fromany class of RNA molecules, found in nature or being preparedsynthetically, and which can induce an immune response. In this context,an immune response may occur in various ways. A substantial factor for asuitable immune response is the stimulation of different T-cellsub-populations. T-lymphocytes are typically divided into twosub-populations, the T-helper 1 (Th1) cells and the T-helper 2 (Th2)cells, with which the immune system is capable of destroyingintracellular (Th1) and extracellular (Th2) pathogens (e.g. antigens).The two Th cell populations differ in the pattern of the effectorproteins (cytokines) produced by them. Thus, Th1 cells assist thecellular immune response by activation of macrophages and cytotoxicT-cells. Th2 cells, on the other hand, promote the humoral immuneresponse by stimulation of the B-cells for conversion into plasma cellsand by formation of antibodies (e.g. against antigens). The Th1/Th2ratio is therefore of great importance in the immune response. Inconnection with the present invention, the Th1/Th2 ratio of the immuneresponse is preferably shifted in the direction towards the cellularresponse (Th1 response) and a cellular immune response is therebyinduced. According to one example, the immune system may be activated byligands of Toll-like receptors (TLRs). TLRs are a family of highlyconserved pattern recognition receptor (PRR) polypeptides that recognizepathogen-associated molecular patterns (PAMPs) and play a critical rolein innate immunity in mammals. Currently at least thirteen familymembers, designated TLR1-TLR13 (Toll-like receptors: TLR1, TLR2, TLR3,TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13), havebeen identified. Furthermore, a number of specific TLR ligands have beenidentified. It was e.g. found that unmethylated bacterial DNA andsynthetic analogs thereof (CpG DNA) are ligands for TLR9 (Hemmi H et al.(2000) Nature 408:740-5; Bauer 5 et al. (2001) Proc NatlAcadSci USA 98,9237-42). Furthermore, it has been reported that ligands for certainTLRs include certain nucleic acid molecules and that certain types ofRNA are immunostimulatory in a sequence-independent orsequence-dependent manner, wherein these various immunostimulatory RNAsmay e.g. stimulate TLR3, TLR7, or TLR8, or intracellular receptors suchas RIG-1, MDA-5, etc. E.g. Lipford et al determined certain G,U-containing oligoribonucleotides as immunostimulatory by acting viaTLR7 and TLR8 (see WO 03/086280). The immunostimulatory G, U-containingoligoribonucleotides described by Lipford et at were believed to bederivable from RNA sources including ribosomal RNA, transfer RNA,messenger RNA, and viral RNA.

According to the present invention, it was found that any RNA (molecule)as e.g. defined above (irrespective of its specific length,strandedness, modification and/or nucleotide sequence) complexed with acarrier peptide according to empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula I) may haveimmunostimulatory properties, i.e. enhance the immune response. RNA asdefined above complexed with a carrier peptide according to empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) (formula I)may thus be used to enhance (unspecific) immunostimulation, if suitableand desired for a specific treatment. Accordingly, it can be anintrinsic property of the complexed RNA of the invention to provideimmunestimulatory effects by complexation of any RNA with a peptideaccording to formula (I).

The at least one (immunostimulatory) RNA (molecule) of the complexed RNAof the present invention may thus comprise any RNA sequence known to beimmunostimulatory, including, without being limited thereto, RNAsequences representing and/or encoding ligands of TLRs, preferablyselected from family members TLR1-TLR13, more preferably from TLR7 andTLR8, ligands for intracellular receptors for RNA (such as RIG-1 orMAD-5, etc.) (see e.g. Meylan, E., Tschopp, J. (2006). Toll-likereceptors and RNA helicases: two parallel ways to trigger antiviralresponses. Mol. Cell 22, 561-569), or any other immunostimulatory RNAsequence. Furthermore, (classes of) RNA molecules, which may be used asimmunostimulatory RNA may include any other RNA capable of eliciting animmune response. Without being limited thereto, such immunostimulatoryRNA may include ribosomal RNA (rRNA), transfer RNA (tRNA), messenger RNA(mRNA), and viral RNA (vRNA).

Such further (classes of) RNA molecules, which may be used as the atleast one (immunostimulatory) RNA (molecule) of the complexed RNA of thepresent invention, may comprise, without being limited thereto, e.g. anRNA molecule of formula (IIa):

G_(l)X_(m)G_(n),

wherein:

-   G is guanosine, uracil or an analogue of guanosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the abovementioned nucleotides;-   l is an integer from 1 to 40,    -   wherein when l=1 G is guanosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are guanosine or an            analogue thereof;-   m is an integer and is at least 3;    -   wherein when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;-   n is an integer from 1 to 40,    -   wherein when n=1 G is guanosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are guanosine or an            analogue thereof.

In addition, such further (classes of) RNA molecules, which may be usedas the at least one (immunostimulatory) RNA (molecule) of the complexedRNA of the present invention may comprise, without being limitedthereto, e.g. an RNA molecule of formula (IIb):

C_(l)X_(m)C_(n),

wherein:

-   C is cytosine, uracil or an analogue of cytosine or uracil;-   X is guanosine, uracil, adenosine, thymidine, cytosine or an    analogue of the abovementioned nucleotides;-   l is an integer from 1 to 40,    -   wherein when l=1 C is cytosine or an analogue thereof,        -   when l>1 at least 50% of the nucleotides are cytosine or an            analogue thereof;-   m is an integer and is at least 3;    -   wherein when m=3 X is uracil or an analogue thereof,        -   when m>3 at least 3 successive uracils or analogues of            uracil occur;-   n is an integer from 1 to 40,    -   wherein when n=1 C is cytosine or an analogue thereof,        -   when n>1 at least 50% of the nucleotides are cytosine or an            analogue thereof.

Preferably, the immunostimulatory RNA molecules as used herein as the atleast one RNA (molecule) of the complexed RNA of the present inventioncomprise a length as defined above in general for RNA molecules of thecomplexed RNA of the present invention, more preferably a length of 5 to5000, of 500 to 5000 or, more preferably, of 1000 to 5000 or,alternatively, of 5 to 1000, 5 to 500, 5 to 250, of 5 to 100, of 5 to 50or, more preferably, of to 30 nucleotides.

The at least one immunostimulatory RNA as used herein as the at leastone RNA (molecule) of the complexed RNA of the present invention may befurthermore modified, preferably “chemically modified” in order toenhance the immunostimulatory properties of said DNA. The term “chemicalmodification” means that the RNA used as immuostimulatory RNA accordingto the invention is modified by replacement, insertion or removal ofindividual or several atoms or atomic groups compared with naturallyoccurring RNA species.

Preferably, the chemical modification of the RNA comprises at least oneanalogue of naturally occurring nucleotides. In a list which is in noway conclusive, examples which may be mentioned for nucleotide analogueswhich can be used according to the invention are analogues of guanosine,uracil, adenosine, thymidine, cytosine. The modifications may refer tomodifications of the base, the ribose moiety and/or the phosphatebackbone moiety. In this context, analogues of guanosine, uracil,adenosine, and cytosine include, without implying any limitation, anynaturally occurring or non-naturally occurring guanosine, uracil,adenosine, thymidine or cytosine that has been altered chemically, forexample by acetylation, methylation, hydroxylation, etc., including1-methyl-adenosine, 1-methyl-guanosine, 1-methyl-inosine,2,2-dimethyl-guanosine, 2,6-diaminopurine, 2′-Amino-2′-deoxyadenosine,2′-Amino-2′-deoxycytidine, 2′-Amino-2′-deoxyguanosine,2′-Amino-2′-deoxyuridine, 2-Amino-6-chloropurineriboside,2-Aminopurine-riboside, 2′-Araadenosine, 2′-Aracytidine, 2′-Arauridine,2′-Azido-2′-deoxyadenosine, 2′-Azido-2′-deoxycytidine,2′-Azido-2′-deoxyguanosine, 2′-Azido-2′-deoxyuridine, 2-Chloroadenosine,2′-Fluoro-2′-deoxyadenosine, 2′-Fluoro-2′-deoxycytidine,2′-Fluoro-2′-deoxyguanosine, 2′-Fluoro-2′-deoxyuridine,2′-Fluorothymidine, 2-methyl-adenosine, 2-methyl-guanosine,2-methyl-thio-N6-isopenenyl-adenosine, 2′-O-Methyl-2-aminoadenosine,2′-O-Methyl-2′-deoxyadenosine, 2′-O-Methyl-2′-deoxycytidine,2′-O-Methyl-2′-deoxyguanosine, 2′-O-Methyl-2′-deoxyuridine,2′-O-Methyl-5-methyluridine, 2′-O-Methylinosine,2′-O-Methylpseudouridine, 2-Thiocytidine, 2-thio-cytosine,3-methyl-cytosine, 4-acetyl-cytosine, 4-Thiouridine,5-(carboxyhydroxymethyl)-uracil, 5,6-Dihydrouridine,5-Aminoallylcytidine, 5-Aminoallyl-deoxy-uridine, 5-Bromouridine,5-carboxymehtylaminomethyl-2-thio-uracil,5-carboxymethylamonomethyl-uracil, 5-Chloro-Ara-cytosine,5-Fluoro-uridine, 5-Iodouridine, 5-methoxycarbonyl methyl-uridine,5-methoxy-uridine, 5-methyl-2-thio-uridine, 6-Azacytidine, 6-Azauridine,6-Chloro-7-deaza-guanosine, 6-Chloropurineriboside,6-Mercapto-guanosine, 6-Methyl-mercaptopurine-riboside,7-Deaza-2′-deoxy-guanosine, 7-Deazaadenosine, 7-methyl-guanosine,8-Azaadenosine, 8-Bromo-adenosine, 8-Bromo-guanosine,8-Mercapto-guanosine, 8-Oxoguanosine, Benzimidazole-riboside,Beta-D-mannosyl-queosine, Dihydro-uracil, Inosine, N1-Methyladenosine,N6-([6-Aminohexyl]carbamoylmethyl)-adenosine, N6-isopentenyl-adenosine,N6-methyl-adenosine, N7-Methyl-xanthosine, N-uracil-5-oxyacetic acidmethyl ester, Puromycin, Queosine, Uracil-5-oxyacetic acid,Uracil-5-oxyacetic acid methyl ester, Wybutoxosine, Xanthosine, andXylo-adenosine. The preparation of such analogues is known to a personskilled in the art, for example from U.S. Pat. No. 4,373,071, U.S. Pat.No. 4,401,796, U.S. Pat. No. 4,415,732, U.S. Pat. No. 4,458,066, U.S.Pat. No. 4,500,707, U.S. Pat. No. 4,668,777, U.S. Pat. No. 4,973,679,U.S. Pat. No. 5,047,524, U.S. Pat. No. 5,132,418, U.S. Pat. No.5,153,319, U.S. Pat. Nos. 5,262,530 and 5,700,642. In the case of ananalogue as described above, particular preference is given according tothe invention to those analogues that increase the immunogenicity of theimmunostimulatory RNA sequence as used herein as the at least one RNA(molecule) of the complexed RNA of the present invention and/or do notinterfere with a further modification that has been introduced into saidimmunostimulatory RNA.

siRNA

In a forth embodiment, the at least one RNA (molecule) of the complexedRNA of the present invention may be in the form of siRNA. A siRNA is ofinterest particularly in connection with the phenomenon of RNAinterference. Attention was drawn to the phenomenon of RNA interferencein the course of immunological research. In recent years, a RNA-baseddefence mechanism has been discovered, which occurs both in the kingdomof the fungi and in the plant and animal kingdom and acts as an “immunesystem of the genome”. The system was originally described in variousspecies independently of one another, first in C. elegans, before it waspossible to identify the underlying mechanisms of the processes as beingidentical: RNA-mediated virus resistance in plants, PTGS(posttranscriptional gene silencing) in plants, and RNA interference ineukaryotes are accordingly based on a common procedure. The in vitrotechnique of RNA interference (RNAi) is based on double-stranded RNAmolecules (dsRNA), which trigger the sequence-specific suppression ofgene expression (Zamore (2001) Nat. Struct. Biol. 9: 746-750; Sharp(2001) Genes Dev. 5:485-490: Hannon (2002) Nature 41: 244-251). In thetransfection of mammalian cells with long dsRNA, the activation ofprotein kinase R and RnaseL brings about unspecific effects, such as,for example, an interferon response (Stark et al. (1998) Annu. Rev.Biochem. 67: 227-264; He and Katze (2002) Viral Immunol. 15: 95-119).These unspecific effects are avoided when shorter, for example 21- to23-mer, so-called siRNA (small interfering RNA), is used, becauseunspecific effects are not triggered by siRNA that is shorter than 30 bp(Elbashir et al. (2001) Nature 411: 494-498). Recently, dsRNA moleculeshave also been used in vivo (McCaffrey et al. (2002), Nature 418: 38-39;Xia et al. (2002), Nature Biotech. 20: 1006-1010; Brummelkamp et al.(2002), Cancer Cell 2: 243-247). Thus, a siRNA as used for the complexedRNA according to the present invention typically comprises a (single-or) double stranded, preferably a double-stranded, RNA sequence withabout 8 to 30 nucleotides, preferably 17 to 25 nucleotides, even morepreferably from 20 to and most preferably from 21 to 23 nucleotides. Inprinciple, all the sections having a length of from 17 to 29, preferablyfrom 19 to 25, most preferably from 21 to 23 base pairs that occur inthe coding region of a RNA sequence as mentioned above, e.g. of an(m)RNA sequence, can serve as target sequence for a siRNA. Equally,siRNAs can also be directed against nucleotide sequences of a(therapeutically relevant) protein or antigen described hereinbeforethat do not lie in the coding region, in particular in the 5′ non-codingregion of the RNA, for example, therefore, against non-coding regions ofthe RNA having a regulatory function. The target sequence of the siRNAcan therefore lie in the translated and/or untranslated region of theRNA and/or in the region of the control elements. The target sequence ofa siRNA can also lie in the overlapping region of untranslated andtranslated sequence; in particular, the target sequence can comprise atleast one nucleotide upstream of the start triplet of the coding regionof the RNA.

Antisense RNA

According to a fifth embodiment, the at least one RNA (molecule) of thecomplexed RNA of the present invention may be an antisense RNA. In thecontext of the present invention, an antisense RNA is preferably a(single-stranded) RNA molecule transcribed on the basis of the coding,rather than the template, strand of DNA, so that it is complementary tothe sense (messenger) RNA. An antisense RNA as used herein as the atleast one RNA (molecule) of the complexed RNA of the present inventiontypically forms a duplex between the sense and antisense RNA moleculesand is thus capable to block translation of the mRNA. An antisense RNAas used herein as the at least one RNA (molecule) of the complexed RNAof the present invention can be directed against (may be complementaryto) any portion of the mRNA sequence, which may encode a(therapeutically relevant) protein or antigen (e.g. as describedhereinbefore), if thereby translation of the encoded protein isreduced/suppressed. Accordingly, the target sequence of the antisenseRNA on the targeted mRNA may be located in the translated and/oruntranslated region of the mRNA, e.g. in the region of the mRNA controlelements, in particular in the 5′ non-coding region of the RNA exertinga regulatory function. The target sequence of an antisense RNA on thetargeted mRNA may also be constructed such that the antisense RNA bindsto the mRNA by covering with its sequence a region which is partiallycomplementary to the untranslated and to translated (coding) sequence ofthe targeted mRNA; in particular, the antisense RNA may be complementaryto the target mRNA sequence by at least one nucleotide upstream of thestart triplet of the coding region of the targeted mRNA. Preferably, theantisense RNA as used herein as the at least one RNA (molecule) of thecomplexed RNA of the present invention comprises a length as generallydefined above for RNA molecules (of the complexed RNA of the presentinvention). Typically the antisense RNA as used herein as the at leastone RNA (molecule) of the complexed RNA of the present invention will bea fragment of the targeted mRNA. In more detail, the antisense RNA mayhave more preferably a length of 5 to 5000, of 500 to 5000, and, morepreferably, of 1000 to 5000 or, alternatively, of 5 to 1000, 5 to 500, 5to 250, of 5 to 100, of 5 to 50 or of 5 to 30 nucleotides, or,alternatively, and even more preferably a length of 20 to 100, of 20 to80, or of 20 to 60 nucleotides.

Modifications of the RNA

According to one embodiment, the RNA as used herein as the at least oneRNA (molecule) of the complexed RNA of the present invention(irrespective of its e.g. specific therapeutic potential, length, and/orsequence), particularly the short RNA oligonucleotide, the coding RNA,the immunostimulatory RNA, the siRNA, the antisense RNA, theriboswitches, ribozymes or aptamers, may provided as a modified RNA,wherein any modification, in particular a modification disclosed in thefollowing) may be introduced (in any combination or as such) into theRNA (molecules) as defined above. Certain types of modifications may,however, be more suitable for specific RNA types (e.g. more suitable forcoding RNA), while other modifications may be applied for any RNAmolecule, e.g. as defined herein without being restricted to specificRNA types. Accordingly, modifications of the RNA may be introduced inorder to achieve specific or complex effects which may desired for theuse of the subject-matter of the invention. Accordingly, modificationsmay be designed to e.g. stabilize the RNA against degradation, toenhance their transfection efficacy, to improve its translationefficacy, to increase their immunogenic potential and/or to enhancetheir therapeutic potential (e.g. enhance their silencing or antisenseproperties). It is particularly preferred, if the modified RNA ascomponent of the inventive complexed RNA allows to combine improvementof at least one, more preferably of at least two functional properties,e.g. to stabilized the RNA and to improve the therapeutic or immunogenicpotential.

Generally, it is a primary object to stabilize the RNA as used herein asthe at least one RNA (molecule) of the complexed RNA of the presentinvention, which allows to extend their half-life time in vivo.Preferably, the half-life time of a modified RNA under in vivoconditions is extended (as compared to the unmodified RNA) by at least20, more preferably at least 40, more preferably at least 50 and evenmore preferably at least 70, 80, 90, 100, 150 or 200%. The stabilizationachieved by the modification may extend the half-life time of themodified mRNA by at least 5, 10, 15, 30 or more preferably at least 60min as compared to the unmodified RNA.

According to one embodiment the at least one RNA (molecule) of thecomplexed RNA of the present invention, preferably a coding RNA, e.g.mRNA, may be stabilized by modifying the G/C content of e.g. the codingregion of the RNA. In a particularly preferred embodiment of the presentinvention, the G/C content of the coding region of the RNA (of thecomplexed RNA of the present invention) is altered, particularlyincreased, compared to the G/C content of the coding region of itscorresponding wild-type RNA, i.e. the unmodified RNA. The encoded aminoacid sequence of the modified RNA is preferably not altered as comparedto the amino acid sequence encoded by the corresponding wild-type RNA.

This modification of the RNA as used herein as the at least one RNA(molecule) of the complexed RNA of the present invention is based on thefact that the coding sequence of any RNA to be translated is importantfor efficient translation of that RNA. In particular, sequences havingan increased G (guanosine)/C (cytosine) content are more stable thansequences having an increased A (adenosine)/U (uracil) content.According to the invention, the codons of the RNA are therefore alteredcompared to the wild-type RNA, while retaining the translated amino acidsequence, such that they include an increased amount of G/C nucleotides.In respect to the fact that several codons code for one and the sameamino acid (so-called degeneration of the genetic code), the mostfavorable codons for the stability can be determined (so-calledalternative codon usage).

Depending on the amino acid to be encoded by the RNA as used herein asthe at least one RNA (molecule) of the complexed RNA of the presentinvention, there are various possibilities for modification of the RNAsequence, compared to its wild-type sequence. In the case of amino acidswhich are encoded by codons which contain exclusively G or Cnucleotides, no modification of the codon is necessary. Thus, the codonsfor Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG) and Gly (GGC orGGG) require no modification, since no A or U is present.

In contrast, codons which contain A and/or U nucleotides can be modifiedby substitution of other codons which code for the same amino acids butcontain no A and/or U. Examples of these are:

the codons for Pro can be modified from CCU or CCA to CCC or CCG;the codons for Arg can be modified from CGU or CGA or AGA or AGG to CGCor CGG;the codons for Ala can be modified from GCU or GCA to GCC or GCG;the codons for Gly can be modified from GGU or GGA to GGC or GGG.

In other cases, although A or U nucleotides cannot be eliminated fromthe codons, it is however possible to decrease the A and U content byusing codons which contain a lower content of A and/or U nucleotides.Examples of these are:

the codons for Phe can be modified from UUU to UUC;the codons for Leu can be modified from UUA, UUG, CUU or CUA to CUC orCUG;the codons for Ser can be modified from UCU or UCA or AGU to UCC, UCG orAGC;the codon for Tyr can be modified from UAU to UAC;the codon for Cys can be modified from UGU to UGC;the codon for His can be modified from CAU to CAC;the codon for Gin can be modified from CAA to CAG;the codons for lie can be modified from AUU or AUA to AUC;the codons for Thr can be modified from ACU or ACA to ACC or ACG;the codon for Asn can be modified from AAU to AAC;the codon for Lys can be modified from AAA to AAG;the codons for Val can be modified from GUU or GUA to GUC or GUG;the codon for Asp can be modified from GAU to GAC;the codon for Glu can be modified from GAA to GAG;the stop codon UAA can be modified to UAG or UGA.

In the case of the codons for Met (AUG) and Trp (UGG), on the otherhand, there is no possibility of sequence modification.

The substitutions listed above can be used either individually or in allpossible combinations to increase the G/C content of the RNA as usedherein as the at least one RNA (molecule) of the complexed RNA of thepresent invention compared to its particular wild-type RNA (i.e. theoriginal sequence). Thus, for example, all codons for Thr occurring inthe wild-type sequence can be modified to ACC (or ACG). Preferably,however, for example, combinations of the above substitutionpossibilities are used:

substitution of all codons coding for Thr in the original sequence(wild-type RNA) to ACC (or ACG) andsubstitution of all codons originally coding for Ser to UCC (or UCG orAGO;substitution of all codons coding for lie in the original sequence toAUC andsubstitution of all codons originally coding for Lys to AAG andsubstitution of all codons originally coding for Tyr to UAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) andsubstitution of all codons originally coding for Glu to GAG andsubstitution of all codons originally coding for Ala to GCC (or GCG) andsubstitution of all codons originally coding for Arg to CGC (or CGG);substitution of all codons coding for Val in the original sequence toGUC (or GUG) andsubstitution of all codons originally coding for Glu to GAG andsubstitution of all codons originally coding for Ala to GCC (or GCG) andsubstitution of all codons originally coding for Gly to GGC (or GGG) andsubstitution of all codons originally coding for Asn to AAC;substitution of all codons coding for Val in the original sequence toGUC (or GUG) andsubstitution of all codons originally coding for Phe to UUC andsubstitution of all codons originally coding for Cys to UGC andsubstitution of all codons originally coding for Leu to CUG (or CUC) andsubstitution of all codons originally coding for Gin to CAG andsubstitution of all codons originally coding for Pro to CCC (or CCG);etc.

Preferably, the G/C content of the coding region of the RNA as usedherein as the at least one RNA (molecule) of the complexed RNA of thepresent invention is increased by at least 7%, more preferably by atleast 15%, particularly preferably by at least 20%, compared to the G/Ccontent of the coded region of the wild-type RNA which codes for aprotein. According to a specific embodiment at least 60%, morepreferably at least 70%, even more preferably at least 80% and mostpreferably at least 90%, 95% or even 100% of the substitutable codons inthe region coding for a protein or the whole sequence of the wild typeRNA sequence are substituted, thereby increasing or even maximizing theGC/content of said sequence.

In this context, it is particularly preferable to increase the G/Ccontent of the RNA as used herein as the at least one RNA (molecule) ofthe complexed RNA of the present invention to the maximum (i.e. 100% ofthe substitutable codons), in particular in the region coding for aprotein, compared to the wild-type sequence.

According to the invention, a further preferred modification of the RNAas used herein as the at least one RNA (molecule) of the complexed RNAof the present invention is based on the finding that the translationefficiency is also determined by a different frequency in the occurrenceof tRNAs in cells. Thus, if so-called “rare codons” are present in anRNA sequence to an increased extent, the corresponding modified RNAsequence is translated to a significantly poorer degree than in the casewhere codons coding for relatively “frequent” tRNAs are present.

According to the invention, in the RNA as used herein as the at leastone RNA (molecule) of the complexed RNA of the present invention, theregion which codes for the protein is modified compared to thecorresponding region of the wild-type RNA such that at least one codonof the wild-type sequence which codes for a tRNA which is relativelyrare in the cell is exchanged for a codon which codes for a tRNA whichis relatively frequent in the cell and carries the same amino acid asthe relatively rare tRNA. By this modification, the RNA sequences aremodified such that codons for which frequently occurring tRNAs areavailable are inserted. In other words, according to the invention, bythis modification all codons of the wild-type sequence which code for atRNA which is relatively rare in the cell can in each case be exchangedfor a codon which codes for a tRNA which is relatively frequent in thecell and which, in each case, carries the same amino acid as therelatively rare tRNA.

Which tRNAs occur relatively frequently in the cell and which, incontrast, occur relatively rarely is known to a person skilled in theart; cf. e.g. Akashi, Curr. Opin. Genet. Dev. 2001, 11(6): 660-666. Thecodons which use for the particular amino acid the tRNA which occurs themost frequently, e.g. the Gly codon, which uses the tRNA which occursthe most frequently in the (human) cell, are particularly preferred.

According to the invention, it is particularly preferable to link thesequential G/C content which is increased, in particular maximized, inthe RNA as used herein as the at least one RNA (molecule) of thecomplexed RNA of the present invention, with the “frequent” codonswithout modifying the amino acid sequence of the protein encoded by thecoding region of the RNA. This preferred embodiment allows provision ofa particularly efficiently translated and stabilized (modified) RNA ofthe complexed RNA according to the present invention.

The determination of the G/C content of an RNA as used herein as the atleast one RNA (molecule) of the complexed RNA of the present invention(increased G/C content; exchange of tRNAs) can be carried out using thecomputer program explained in WO 02/098443—the disclosure content ofwhich is included in its full scope in the present invention. Using thiscomputer program, the nucleotide sequence of any desired RNA can bemodified with the aid of the genetic code or the degenerative naturethereof such that a maximum G/C content results, in combination with theuse of codons which code for tRNAs occurring as frequently as possiblein the cell, the amino acid sequence coded by the RNA (molecule)preferably not being modified compared to the non-modified sequence.Alternatively, it is also possible to modify only the G/C content oronly the codon usage compared to the original sequence. The source codein Visual Basic 6.0 (development environment used: Microsoft VisualStudio Enterprise 6.0 with Servicepack 3) is also described in WO02/098443.

In a further preferred embodiment of the present invention, the A/Ucontent in the environment of the ribosome binding site of the at leastone RNA (molecule) of the complexed RNA of the present invention isincreased compared to the A/U content in the environment of the ribosomebinding site of its particular wild-type RNA. This modification (anincreased A/U content around the ribosome binding site) increases theefficiency of ribosome binding to the modified RNA. An effective bindingof the ribosomes to the ribosome binding site (Kozak sequence:GCCGCCACCAUGG (SEQ ID NO: 33), the AUG forms the start codon) in turnhas the effect of an efficient translation of the modified RNA.

According to a further embodiment of the present invention the at leastone RNA (molecule) of the complexed RNA of the present invention may bemodified with respect to potentially destabilizing sequence elements.Particularly, the coding region and/or the 5′ and/or 3′ untranslatedregion of this RNA may be modified compared to the particular wild-typeRNA such that is contains no destabilizing sequence elements, the codedamino acid sequence of the RNA (molecule) preferably not being modifiedcompared to its particular wild-type RNA. It is known that, for example,in sequences of eukaryotic RNAs destabilizing sequence elements (DSE)occur, to which signal proteins bind and regulate enzymatic degradationof RNA in vivo. For further stabilization of the RNA (molecule),optionally in the region which encodes for a protein, one or more suchmodifications compared to the corresponding region of the wild-type RNAcan therefore be carried out, so that no or substantially nodestabilizing sequence elements are contained there. According to theinvention, DSE present in the untranslated regions (3′- and/or 5′-UTR)can also be eliminated from the at least one RNA (molecule) of thecomplexed RNA of the present invention by such modifications.

Such destabilizing sequences are e.g. AU-rich sequences (AURES), whichoccur in 3′-UTR sections of numerous unstable RNAs (Caput et at, Proc.Natl. Acad. Sci. USA 1986, 83: 1670 to 1674). The RNA of the complexedRNA according to the present invention is therefore preferably modifiedcompared to the wild-type RNA such that the RNA contains no suchdestabilizing sequences. This also applies to those sequence motifswhich are recognized by possible endonucleases, e.g. the sequenceGAACAAG, which is contained in the 3′-UTR segment of the gene whichcodes for the transferrin receptor (Binder et al., EMBO J. 1994, 13:1969 to 1980). These sequence motifs are also preferably removedaccording to the invention in the at least one RNA (molecule) of thecomplexed RNA of the present invention.

According to the present invention, the at least one RNA (molecule) ofthe complexed RNA of the present invention can have a 5′ cap structure.Examples of cap structures which can be used according to the inventionare m7G(5′)ppp, (5′(A,G(5′)ppp(5′)A and G(5′)ppp(5′)G.

Another modification enhancing the stability of the at least one RNA(molecule) of the complexed RNA of the present invention is based on 5′-or 3′ elongations of the RNA, typically homonucleotide elongations of alength of 10 to 200 nucleotides. These elongations may contain,particularly if the RNA is provided as mRNA, a poly-A tail at the 3′terminus of typically about 10 to 200 adenosine nucleotides, preferablyabout 10 to 100 adenosine nucleotides, more preferably about 20 to 70adenosine nucleotides or even more preferably about 20 to 60 adenosinenucleotides. Alternatively or additionally, the at least one RNA(molecule) of the complexed RNA of the present invention may contain,particularly if the RNA is provided as mRNA, a poly-C tail at the 3′terminus of typically about 10 to 200 cytosine nucleotides, preferablyabout 10 to 100 cytosine nucleotides, more preferably about 20 to 70cytosine nucleotides or even more preferably about 20 to 60 or even 10to 40 cytosine nucleotides.

Another modification, which may occur in the at least one RNA (molecule)of the complexed RNA of the present invention, particularly if the RNAis provided as mRNA, refers preferably to at least one IRES and/or atleast one 5′ and/or 3′ stabilizing sequence. According to the invention,one or more so-called IRES (internal ribosomal entry site) may beinserted into the RNA. An IRES can thus function as the sole ribosomebinding site, but it can also serve to provide a RNA which codes severalproteins which are to be translated by the ribosomes independently ofone another (multicistronic RNA). Examples of IRES sequences which canbe used according to the invention are those from picornaviruses (e.g.FMDV), pestiviruses (CFFV), polioviruses (PV), encephalomyocarditisviruses (ECMV), foot and mouth disease viruses (FMDV), hepatitis Cviruses (HCV), classical swine fever viruses (CSFV), mouse leukoma virus(MLV), simian immunodeficiency viruses (SIV) or cricket paralysisviruses (CrPV).

According to the invention, the at least one RNA (molecule) of thecomplexed RNA of the present invention may exhibit at least one 5′and/or 3′ stabilizing sequence as known from the art. These stabilizingsequences in the 5′ and/or 3′ untranslated regions have the effect ofincreasing the half-life of the RNA in the cytosol. These stabilizingsequences can have 100% sequence homology to naturally occurringsequences which occur in viruses, bacteria and eukaryotes, but can alsobe partly or completely synthetic. The untranslated sequences (UTR) ofthe globin gene, e.g. from Homo sapiens or Xenopus laevis may bementioned as an example of stabilizing sequences which can be used inthe present invention for a stabilized RNA. Another example of astabilizing sequence has the general formula(C/U)CCAN_(x)CCC(U/A)Py_(x)UC(C/U)CC (SEQ ID NO: 34), which is containedin the 3′UTR of the very stable RNA which codes for globin,(I)-collagen, 15-lipoxygenase or for tyrosine hydroxylase (cf. Holcik etat, Proc. Natl. Acad. Sci. USA 1997, 94: 2410 to 2414). Such stabilizingsequences can of course be used individually or in combination with oneanother and also in combination with other stabilizing sequences knownto a person skilled in the art. The at least one RNA (molecule) of thecomplexed RNA of the present invention is therefore preferably presentas globin UTR (untranslated regions)-stabilized RNA, in particular asglobin UTR-stabilized RNA.

If desired, the at least one RNA (molecule) of the complexed RNA of thepresent invention may contain backbone modifications. A backbonemodification in connection with the present invention is a modificationin which phosphates of the backbone of the nucleotides contained in theRNA are chemically modified. Such backbone modifications typicallyinclude, without implying any limitation, modifications from the groupconsisting of methylphosphonates, phosphoramidates and phosphorothioates(e.g. cytidine-5′-O-(1-thiophosphate)).

The at least one RNA (molecule) of the complexed RNA of the presentinvention may additionally or alternatively also contain sugarmodifications. A sugar modification in connection with the presentinvention is a chemical modification of the sugar of the nucleotidespresent and typically includes, without implying any limitation, sugarmodifications selected from the group consisting of2′-deoxy-2′-fluoro-oligoribonucleotide(2′-fluoro-2′-deoxycytidine-5′-triphosphate,2′-fluoro-2′-deoxyuridine-5′-triphosphate), 2′-deoxy-2′-deamineoligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate,2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide(2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5′-triphosphate),2′-C-alkyl oligoribonucleotide, and isomers thereof(2′-aracytidine-5′-triphosphate, 2′-arauridine-5′-triphosphate), orazidotriphosphate (2′-azido-2′-deoxycytidine-5′-triphosphate,2′-azido-2′-deoxyuridine-5′-triphosphate).

The at least one RNA (molecule) of the complexed RNA of the presentinvention may additionally or alternatively also contain at least onebase modification, which is preferably suitable for increasing theexpression of the protein coded for by the at least one RNA (molecule)significantly as compared with the unaltered, i.e. natural (=native),RNA sequence. Significant in this case means an increase in theexpression of the protein compared with the expression of the native RNAsequence by at least 20%, preferably at least 30%, 40%, 50% or 60%, morepreferably by at least 70%, 80%, 90% or even 100% and most preferably byat least 150%, 200% or even 300%. In connection with the presentinvention, a nucleotide having a base modification is preferablyselected from the group of the base-modified nucleotides consisting of2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate. Particular preference is given tonucleotides for base modifications selected from the group ofbase-modified nucleotides consisting of5-methylcytidine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,5-bromocytidine-5′-triphosphate, and pseudouridine-5′-triphosphate.

The at least one RNA (molecule) of the complexed RNA of the presentinvention may additionally or alternatively also contain at least onemodification of a nucleoside of a nucleotide as contained in the atleast one RNA (molecule), which acts immunosuppressive, i.e. ispreferably suitable for preventing or decreasing an immune response,when administered to a patient in need thereof. Such at least onemodification is preferably selected from nucleoside modificationsselected from:

-   -   a) a chemical modification at the 4-, 5- or 6-position of the        pyrimidine base of the nucleosides of cytidine and/or uridine;    -   b) a chemical modification at the 2-, 6-, 7- or 8-position of        the purine base of the nucleosides of adenosine, inosine and/or        guanosine; and/or    -   c) a chemical modification at the 2′-position of the sugar of        the nucleosides of adenosine, inosine, guanosine, cytidine        and/or uridine.

In this context, an (m)RNA is a nucleic acid chain formed by a number ofnucleotides typically selected from adenosine-5′-monophosphate,guanosine-5′-monophosphate, inosine-5′-monophosphate,cytidine-5′-monophosphate and/or uridine-5′-monophosphate. Thosenucleotides are linked to each other via their monophosphate.Nucleotides comprise nucleosides and a 5′-monophosphate as a structuralcomponent, wherein the nucleosides are typically formed by a nucleobase,i.e. a pyrimidine (uracil or cytosine) or a purine (adenine or guanine)base, and a sugar. Accordingly, a modification of a nucleoside of atleast one RNA (molecule) of the complexed RNA of the present inventionis always intended to mean a modification in the nucleoside structure ofthe respective nucleotide of said at least one RNA (molecule).

According to a first modification a), at least one nucleoside of the atleast one RNA (molecule) of the complexed RNA of the present invention,may be modified with a chemical modification at the 5- or 6-position ofthe pyrimidine base of the nucleosides cytidine and/or uridine. Withoutbeing limited thereto, such chemical modifications at the 4-, 5- or6-position of the base pyrimidine of the nucleosides cytidine and/oruridine may be selected from the group consisting of: 4-thio,5-iodo-/(5-I—), 5-bromo-/(5-Br—), 5-aminoallyl-, 5-fluoro-/(5-F—),5-hydroxy-, 5-hydro-/(5-H—), 5-nitro-, 5-propynyl-/(5-(C≡C—CH₃)—),5-methyl-, 5-methyl-2-thio-, 5-formyl-, 5-hydroxymethyl-, 5-methoxy-,5-oxyacetic acid methyl ester-, 5-oxyacetic acid-,5-carboxyhydroxymethyl-, 5-(carboxyhydroxymethyl)pyrimidine methylester-, 5-methoxycarbonylmethyl-, 5-methoxycarbonylmethyl-2-thio,5-aminomethyl-, 5-aminomethyl-2-thio-, 5-aminomethyl-2-seleno-,5-methylaminomethyl-, 5-carbamoylmethyl-, 5-carboxymethylaminomethyl-,5-carboxymethylaminomethyl-2-thio-, 5-carboxymethyl-, 5-methyldihydro-,5-taurinomethyl-, 5-taurinomethyl-2-thiouridine,5-isopentenylaminomethyl-, 5-isopentenylaminomethyl-2-thio-,5-aminopropyl-/(5-(C₃H₆NH₃)—),5-methoxy-ethoxy-methyl-/(5-(CH₂—O—C₂H₄—O—CH₃)—), or 6-aza-.

According to second modification b), at least one nucleoside of the atleast one RNA (molecule) of the complexed RNA of the present invention,suitable for suppressing and/or avoiding an (innate) immunostimulatoryresponse in a mammal typically exhibited when administering thecorresponding unmodified at least one RNA (molecule), may bealternatively modified with a chemical modification at the 2-, 6-, 7- or8-position of the purine base of the nucleosides adenosine, inosineand/or guanosine. Without being limited thereto, such chemicalmodifications at the 2-, 6-, 7- or 8-position of the purine base of thenucleosides adenosine, inosine and/or guanosine may be selected from thegroup consisting of 2-Amino-, 7-Deaza-, 8-Aza-, or 8-Azido-.

According to a third modification c), at least one nucleoside of the atleast one RNA (molecule) of the complexed RNA of the present invention,suitable for suppressing and/or avoiding an (innate) immunostimulatoryresponse in a mammal typically exhibited when administering thecorresponding unmodified at least one RNA (molecule), may be modifiedwith at least one chemical modification at the 2′-position of the sugarof the nucleosides adenosine, inosine, guanosine, cytidine and/oruridine, when incorporated in the RNA sequence. Without being limitedthereto, such chemical modifications at the 2′-position of the sugar ofthe nucleosides adenosine, inosine, guanosine, cytidine and/or uridinemay be selected from the group consisting of: 2′-deoxy-,2′-amino-2′-deoxy-, 2′-amino-, 2′-fluoro-2′-deoxy-, 2′-fluoro-,2′-O-methyl-2′-deoxy- or 2′-O-methyl-.

According to a particularly preferred embodiment, at least onenucleoside of the at least one RNA (molecule) of the complexed RNA ofthe present invention has been modified at the 4-, 5- or 6-position ofthe base pyrimidine of the nucleosides cytidine and/or uridine and atthe 2′-position of the ribose sugar according to modifications a) and c)as defined above.

According to another particularly preferred embodiment, at least onenucleoside of the at least one RNA (molecule) of the complexed RNA ofthe present invention has been modified at the 2-, 6-, 7- or 8-positionof the purine base of the nucleosides adenosine, inosine and/orguanosine and at the 2′-position of the ribose sugar according tomodifications b) and c) as defined above, more preferably as definedabove.

According to an even more particularly preferred embodiment, at leastone nucleoside of the at least one RNA (molecule) of the complexed RNAof the present invention has been modified leading to chemicallymodified nucleotides (of the (m)RNA) selected from the following group:4-thio-uridine-5′-(mono)phosphate,2-Aminopurine-riboside-5′-(mono)phosphate,5-Aminoallylcytidine-5′-(mono)phosphate,5-Aminoallyluridine-5′-(mono)phosphate,5-Bromocytidine-5′-(mono)phosphate,5-Bromo-2′-deoxycytidine-5′-(mono)phosphate,5-Bromouridine-5′-(mono)phosphate,5-Bromo-2′-deoxyuridine-5′-(mono)phosphate,5-Iodocytidine-5′-(mono)phosphate,5-Iodo-2′-deoxycytidine-5′-(mono)phosphate,5-Iodouridine-5′-(mono)phosphate,5-Iodo-2′-deoxyuridine-5′-(mono)phosphate,5-Propynyl-2′-deoxycytidine-5′-(mono)phosphate,5-Propynyl-2′-deoxyuridine-5′-(mono)phosphate,5-formylcytidine-5′-(mono)phosphate,5,2′-O-dimethylcytidine-5′-(mono)phosphate,5-hydroxymethylcytidine-5′-(mono)phosphate,5-formyl-2′-O-methylcytidine-5′-(mono)phosphate,5,2′-O-dimethyluridine-5′-(mono)phosphate,5-methyl-2-thiouridine-5′-(mono)phosphate,5-hydroxyuridine-5′-(mono)phosphate,5-methoxyuridine-5′-(mono)phosphate, uridine 5-oxyaceticacid-5′-(mono)phosphate, uridine 5-oxyacetic acid methylester-5′-(mono)phosphate,5-(carboxyhydroxymethyl)uridine-5′-(mono)phosphate,5-(carboxyhydroxymethyl)uridine methyl ester-5′-(mono)phosphate,5-methoxycarbonylmethyluridine-5′-(mono)phosphate,5-methoxycarbonylmethyl-2′-O-methyluridine-5′-(mono)phosphate,5-methoxycarbonylmethyl-2-thiouridine-5′-(mono)phosphate,5-aminomethyl-2-thiouridine-5′-(mono)phosphate,5-methylaminomethyluridine-5′-(mono)phosphate,5-methylaminomethyl-2-thiouridine-5′-(mono)phosphate,5-methylaminomethyl-2-selenouridine-5′-(mono)phosphate,5-carbamoylmethyluridine-5′-(mono)phosphate,5-carbamoylmethyl-2′-O-methyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyl-2′-O-methyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyl-2-thiouridine-5′-(mono)phosphate,5-carboxymethyluridine-5′-(mono)phosphate,5-methyldihydrouridine-5′-(mono)phosphate,5-taurinomethyluridine-5′-(mono)phosphate,5-taurinomethyl-2-thiouridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)uridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)-2-thiouridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)-2′-O-methyluridine-5′-(mono)phosphate,6-Azacytidine-5′-(mono)phosphate, 7-Deazaadenosine-5′-(mono)phosphate,7-Deazaguanosine-5′-(mono)phosphate, 8-Azaadenosine-5′-(mono)phosphate,8-Azidoadenosine-5′-(mono)phosphate, Pseudouridine-5′-(mono)phosphate,2′-Amino-2′-deoxycytidine-(mono)phosphate,2′-Fluorothymidine-5′-(mono)phosphate, inosine-5′-(mono)phosphate,2′-O-Methyl-inosine-5′-(mono)phosphate.

If desired, the at least one RNA (molecule) of the complexed RNA of thepresent invention may contain substitutions, additions or deletions ofnucleotides, which are preferably introduced to achieve functionaleffects. These various types of nucleotide modifications may beintroduced, if the RNA, e.g the mRNA, is derived from a WT sequence.Hereby, a DNA matrix is used for preparation of the RNA of the complexedRNA according to the present invention by techniques of the well knownsite directed mutagenesis (see e.g. Maniatis et al., Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., ColdSpring Harbor, N.Y., 2001). In such a process, for preparation of theRNA, a corresponding DNA molecule may be transcribed in vitro. This DNAmatrix has a suitable promoter, e.g. a T7 or SP6 promoter, for in vitrotranscription, which is followed by the desired nucleotide sequence forthe RNA to be prepared and a termination signal for in vitrotranscription. According to the invention, the DNA molecule which formsthe matrix of a RNA of interest may be prepared by fermentativeproliferation and subsequent isolation as part of a plasmid which can bereplicated in bacteria. Plasmids which may be mentioned as suitable forthe present invention are e.g. the plasmids pT7 Ts (GenBank accessionnumber U26404; Lai et al., Development 1995, 121: 2349 to 2360), pGEM@series, e.g. pGEM®-1 (GenBank accession number X65300; from Promega) andpSP64 (GenBank accession number X65327); cf. also Mezei and Storts,Purification of PCR Products, in: Griffin and Griffin (ed.), PCRTechnology: Current Innovation, CRC Press, Boca Raton, Fla., 2001.

The mass or molar ratio of the components of the RNA complex accordingto the present invention, which means the mass or molar ratio of the RNA(be it single- or double-stranded) to the one or more oligopeptidestypically is by no way restricted and is chosen as suitable for theparticular application. However, the mass or molar ratio of the one ormore oligopeptides and the RNA may be less than 1:1, 1:2, 1:3, 1:4, 1:5,1:6, 1:7, 1:8, 1:9, 1:10, 1:11, 1:12, 1:13, 1:14, 1:15, 1:16, 1:17,1:18, or less than 1:20. Alternatively, the mass or molar ratio of theone or more oligopeptides and the RNA may higher than 1:1, 2:1, 3:1,4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 11:1, 12:1, 13:1, 14:1, 15:1, 16:1,17:1, 18:1, 19:1, 20:1. Preferably, the mass or molar ratio of the oneor more oligopeptides and the RNA may not be less than 1:5 with respectto the content of the one or more oligopeptides. More preferably, the(molar or) mass ratio of the one or more oligopeptides and the RNA isfrom 1:5 to 20:1, more preferably from 1:3 to 15:1.

According to a particular preferred embodiment, the mass ratio of thecomponents of the RNA complex according to the present invention,particularly the mass ratio of the at least one RNA (molecule) of thecomplexed RNA to the one or more oligopeptides is preferably in a rangeof about 1:100 to about 1:0.5, more preferably has a value of about 1:50to about 1:1, or even more preferably about 1:100, about 1:90, about1:80, about 1:70, about 1:60, about 1:50, about 1:45, about 1:40, about1:35, about 1:30, about 1:25, about 1:20, about 1:15, about 1:10, about1:5, about 1:4, about 1:3, about 1:2, about 1:1 or even about 1:0.5regarding the ratio of RNA:peptide in the complex, wherein any range maybe formed by combining two of the above specifically indicated values.Most preferably, the mass ratio of the at least one RNA (molecule) ofthe complexed RNA to the one or more oligopeptides may be in a range ofabout 1:50 to about 1:1.

Likewise, the molar ratio of the components of the RNA complex accordingto the present invention, particularly the molar ratio of the at leastone RNA (molecule) of the complexed RNA to the one or more oligopeptidesis preferably, according to a particular preferred embodiment, in arange of about 1:20000 to about 1:500 or even 1:250, more preferably ina range of about 1:10000 to about 1:1000, or even more preferably has avalue of about 1:9500, about 1:9000, about 1:8500, about 1:8000, about1:7500, about 1:7000, about 1:6500, about 1:6000, about 1:5500, about1:5000, about 1:4500, about 1:4000, about 1:3500, about 1:3000, about1:2500, about 1:2000, about 1:1500, about 1:1000, about 1:500, about1:450, about 1:400, about 1:350, about 1:300, or about 1:250 regardingthe ratio of RNA:peptide in the complex, wherein any range may be formedby combining two of the above specifically indicated values. Mostpreferably, the molar ratio of the at least one RNA (molecule) of thecomplexed RNA to the one or more oligopeptides may be in a range ofabout 1:10000 to about 1:1000. For immunostimulation purposes, the molarratio of the components of the RNA complex according to the presentinvention may be in a range of about 1:10000 to about 1:100 or even in arange of about 1:10000 to about 1:500.

In the context of the present invention, the molar ratio and the massratio are typically dependent on each other, wherein each of theseratios may be influenced by factors such as RNA length or peptidelength. However, for purposes of determination, the mass ratio and themolar ratio may be calculated for an average complex size, wherein amass ratio of about 1:50-1:1 approximately corresponds to a molar ratioof about 1:10000-1:1000. An exemplary schedule of molar and mass ratiosis given in the Examples, which may be used for calculationadditionally.

Furthermore, the ratio of the RNA complex components according to thepresent invention, particularly the ratio of the at least one RNA(molecule) of the complexed RNA to the one or more oligopeptides, mayalso be calculated on the basis of the nitrogen/phosphate ratio(N/P-ratio) of the entire RNA complex. For example, 1 μg RNA typicallycontains about 3 nmol phosphate residues, provided the RNA exhibits astatistical distribution of bases. Additionally, 1 μg peptide typicallycontains about x nmol nitrogen residues, dependent on the molecularweight and the number of basic amino acids. When exemplarily calculatedfor (Arg)₉ (molecular weight 1424 g/mol, 9 nitrogen atoms), 1 μg (Arg)₉contains about 700 pmol (Arg)₉ and thus 700×9=6300 pmol basic aminoacids=6.3 nmol nitrogen atoms. For a mass ratio of about 1:1 RNA/(Arg)₉an N/P ratio of about 2 can be calculated. When exemplarily calculatedfor protamine (molecular weight about 4250 g/mol, 21 nitrogen atoms,when protamine from salmon is used) with a mass ratio of about 2:1 with2 μg RNA, 6 nmol phosphate are to be calculated for the RNA; 1 μgprotamine contains about 235 pmol protamine molecules and thus235×21=4935 pmol basic nitrogen atoms=4.9 nmol nitrogen atoms. For amass ratio of about 2:1 RNA/protamine an N/P ratio of about 0.81 can becalculated. For a mass ratio of about 8:1 RNA/protamine an N/P ratio ofabout 0.2 can be calculated. In the context of the present invention, anN/P-ratio is preferably in the range of about 0.2-50, preferably in arange of about 0.5-50 and most preferably in a range of about 0.75-25 or1-25 regarding the ratio of RNA:peptide in the complex, even morepreferably in the range of about 10-50 and most preferably in the rangeof about 25-50).

Another embodiment of the present invention relates to a composition,preferably a pharmaceutical composition, comprising a complexed RNAaccording to the present invention and optionally a (pharmaceutically)suitable carrier and/or further auxiliary substances and additives. The(pharmaceutical) composition employed according to the present inventiontypically comprises a safe and effective amount of a complexed RNAaccording to the present invention. As used herein, a “safe andeffective amount” means an amount of a complexed RNA according to thepresent invention such as to provide an effect in cells or tissues invitro or in vivo, e.g. to induce significantly an expression (in vitroor in vivo) of an encoded protein as described above, such as atherapeutically active protein, an antibody or an antigene, or any otherprotein or peptide as described above, to induce a positive change of astate to be treated (in vivo) in a cell, a tissue or an organism, e.g. atumour disease or cancer disease, a cardiovascular disease, aninfectious disease, an autoimmune disease, (mono-)genetic diseases, etc.as described herein, and/or to induce or enhance an immune response. Atthe same time, however, a “safe and effective amount” is low enough toavoid serious side effects, particularly in the therapy of diseases asmentioned herein, that is to say to render possible a reasonable ratioof advantage and risk. Determination of these limits typically lieswithin the range of reasonable medical judgement. The concentration ofthe complexed RNA according to the invention in such (pharmaceutical)compositions can therefore vary, for example, without being limitedthereto, within a wide range of from e.g. 0.1 ng to 1,000 mg/ml or evenmore. Such a “safe and effective amount” of a complexed RNA according tothe invention can vary in connection with the particular state to betreated and the age and the physical state of the patient to be treated,the severity of the state, the duration of the treatment, the nature ofthe concomitant therapy, of the particular (pharmaceutically) suitablecarrier used and similar factors within the knowledge and experience ofthe treating doctor. The (pharmaceutical) composition described here canbe employed for human and also for veterinary medicine purposes.

The (pharmaceutical) composition according to the invention describedhere can optionally comprise a suitable carrier, preferably apharmaceutically suitable carrier. The term “suitable carrier” used herepreferably includes one or more compatible solid or liquid fillers, ordiluents or encapsulating compounds which are suitable foradministration to a person. The term “compatible” as used here meansthat the constituents of the composition are capable of being mixedtogether with the complexed RNA according to the invention and theauxiliary substance optionally contained in the composition, as such andwith one another in a manner such that no interaction which wouldsubstantially reduce the (pharmaceutical) effectiveness of thecomposition under usual condition of use occurs, such as e.g. wouldreduce the (pharmaceutical) activity of the encoded proteins or evensuppress or impair expression of the coded proteins or e.g. wouldinhibit the immunogenic potential of the complexed RNA. Suitable carriermust of course have a sufficiently high purity and a sufficiently lowtoxicity to render them suitable for administration to a person to betreated.

Carriers are chosen dependent on the way of administration, be it insolid or liquid form. Accordingly, the choice of a (pharmaceutically)suitable carrier as described above is determined in particular by themode in which the (pharmaceutical) composition according to theinvention is administered. The (pharmaceutical) composition according tothe invention can be administered, for example, systemically.Administration routes include e.g. intra- or transdermal, oral,parenteral, including subcutaneous, intramuscular, i.a. or intravenousinjections, topical and/or intranasal routes. The suitable amount of the(pharmaceutical) composition according to the invention which is to beused can be determined by routine experiments using animal models. Suchmodels include, but without being limited thereto, models of the rabbit,sheep, mouse, rat, dog and non-human primate models.

If administered in liquid form, e.g. by injection, the carrier may beselected from pyrogen-free water; isotonic saline solution and bufferedsolutions, e.g. phosphate buffered solutions. Preferred unit dose formsfor injection include sterile solutions of water, physiological salinesolution or mixtures thereof, e.g. Ringer-Lactat solution. The pH ofsuch solutions should be adjusted to about 7.0 to about 7.6, preferablyabout 7.4.

Preferably, the (pharmaceutical) composition contains the inventivecomplexed RNA in water. Alternatively, the (pharmaceutical) compositionaccording to the invention may contain an injection buffer as carrierfor liquid preparation, which preferably improves transfection and, ifthe RNA of the complexed RNA of the present invention codes for aprotein, also the translation of the encoded protein, in cells, tissuesor an organism. The (pharmaceutical) composition according to theinvention can comprise, for example, an aqueous injection buffer orwater which contains, with respect to the total (pharmaceutical)composition, if this is in liquid form, a sodium salt, preferably atleast 50 mM sodium salt, a calcium salt, preferably at least 0.01 mMcalcium and/or magnesium salt, and optionally a potassium salt,preferably at least 3 mM potassium salt. According to a preferredembodiment, the sodium salts, calcium and/or magnesium salts andoptionally potassium salts contained in such an injection buffer are inthe form of halides, e.g. chlorides, iodides or bromides, or in the formof their hydroxides, carbonates, bicarbonates or sulfates. Exampleswhich are to be mentioned here are, for the sodium salt NaCl, NaI, NaBr,Na₂CO₃, NaHCO₃, and/or Na₂SO₄, for the potassium salt optionally presentKCl, KI, KBr, K₂CO₃, KHCO₃, and/or K₂SO₄, and for the calcium and/ormagnesium salt CaCl, CaI₂, CaBr₂, CaCO₃, CaSO₄, Ca(OH)₂, MgCl₂, MgI₂,MgBr₂, MgCO₃, MgSO₄, and/or Mg(OH)₂. The injection buffer can alsocontain organic anions of the abovementioned cations. In a particularlypreferred embodiment, such an injection buffer contains as salts sodiumchloride (NaCl), calcium chloride (CaCl₂) and optionally potassiumchloride (KCl), it also being possible for other anions to be present inaddition to the chlorides. These salts are typically present in theinjection buffer optionally used in the (pharmaceutical) compositionaccording to the invention, with respect to the total (pharmaceutical)composition (if this is in liquid form), in a concentration of at least50 mM sodium chloride (NaCl), at least 3 mM potassium chloride (KCl) andat least 0.01 mM calcium and/or magnesium chloride (CaCl₂). Theinjection buffer can be in the form of both hypertonic and isotonic orhypotonic injection buffers. In connection with the present invention,in this context the injection buffer is hypertonic, isotonic orhypotonic in each case with respect to the particular reference medium,i.e. the injection buffer has either a higher, the same or a lower saltcontent compared with the particular reference medium, suchconcentrations of the abovementioned salts which do not lead to damageto the cells caused by osmosis or other concentration effects preferablybeing employed. Reference media here are, for example, liquids whichoccur in “in vivo” methods, such as, for example, blood, lymph fluid,cytosol fluids or other fluids which occur in the body, or liquids orbuffers conventionally employed in “in vitro” methods. Such liquids andbuffers are known to a person skilled in the art.

The injection buffer optionally contained in the (pharmaceutical)composition according to the invention can also contain furthercomponents, for example sugars (mono-, di-, tri- or polysaccharides), inparticular glucose or mannitol. In a preferred embodiment, however, nosugars are present in the injection buffer used. It is also preferablefor the injection buffer precisely to contain no non-charged components,such as, for example, sugars. The injection buffer typically containsexclusively metal cations, in particular from the group consisting ofthe alkali or alkaline earth metals, and anions, in particular theanions described above. The pH of the injection buffer used, withrespect to the total (pharmaceutical) composition, if this is in liquidform, is preferably between 1 and 8.5, preferably between 3 and 5, morepreferably between 5.5 and 7.5, in particular between 5.5 and 6.5. Ifappropriate, the injection buffer can also contain a buffer system whichfixes the injection buffer at a buffered pH. This can be, for example, aphosphate buffer system, HEPES or Na₂HPO₄/NaH₂PO₄. However, theinjection buffer used very particularly preferably contains none of theabovementioned buffer systems or contains no buffer system at all.

The injection buffer optionally contained in the (pharmaceutical)composition according to the invention can contain, in addition to or asan alternative to the monovalent and divalent cations described,divalent cations, in particular from the group consisting of alkalineearth metals, such as, for example, magnesium (Mg²⁺), or also iron(Fe²⁺), and monovalent cations, in particular from the groups consistingof alkali metals, such as, for example, lithium (Li⁺). These monovalentcations are preferably in the form of their salts, e.g. in the form ofhalides, e.g. chlorides, iodides or bromides, or in the form of theirhydroxides, carbonates, bicarbonates or sulfates. Examples which are tobe mentioned here are, for the lithium salt LiCl, LiI, LiBr, Li₂CO,LiHCO₃, Li₂SO₄, for the magnesium salt MgCl₂, MgI₂, MgBr₂, MgCO₃, MgSO₄,and Mg(OH)₂, and for the iron salt FeCl₂, FeBr₂, FeI₂, FeF₂, Fe₂O₃,FeCO₃, FeSO₄, Fe(OH)₂. All the combinations of di- and/or monovalentcations, as described above, are likewise included. Such injectionbuffers which contain only divalent, only monovalent or di- andmonovalent cations can thus be used in the (pharmaceutical) compositionaccording to the invention. Such injection buffers which contain onlyone type of di- or monovalent cations, particularly preferably e.g. onlyCa²⁺ cations, or a salt thereof, e.g. CaCl₂, can likewise be used. Themolarities given above for Ca²⁺ (as a divalent cation) and Na¹⁺ (as amonovalent cation) (that is to say typically concentrations of at least50 mM Na⁺, at least 0.01 mM Ca²⁺ and optionally at least 3 mM K⁺) in theinjection buffer can also be taken into consideration if another di- ormonovalent cation, in particular other cations from the group consistingof the alkaline earth metals and alkali metals, are employed instead ofsome or all the Ca²⁺ or, respectively, Na¹⁺ in the injection buffer usedaccording to the invention for the preparation of the injectionsolution. All the Ca²⁺ or Na¹⁺, as mentioned above, can indeed bereplaced by in each case other di- or, respectively, monovalent cationsin the injection buffer used, for example also by a combination of otherdivalent cations (instead of Ca²⁺) and/or a combination of othermonovalent cations (instead of Na¹⁺) (in particular a combination ofother divalent cations from the group consisting of the alkaline earthmetals or, respectively, of other monovalent cations from the groupconsisting of the alkali metals), but it is preferable to replace atmost some of the Ca²⁺ or Na⁺, i.e. for at least 20%, preferably at least40%, even more preferably at least 60% and still more preferably atleast 80% of the particular total molarities of the mono- and divalentcations in the injection to be occupied by Ca²⁺ and, respectively, Na¹⁺.However, it is very particularly preferable if the injection bufferoptionally contained in the pharmaceutical composition according to theinvention contains exclusively Ca¹⁺ as a divalent cation and Na¹⁺ as amonovalent cation, that is to say, with respect to the totalpharmaceutical composition, Ca²⁺ represents 100% of the total molarityof divalent cations, just as Na¹⁺ represents 100% of the total molarityof monovalent cations. The aqueous solution of the injection buffer cancontain, with respect to the total pharmaceutical composition, up to 30mol % of the salts contained in the solution, preferably up to 25 mol %,preferably up to 20 mol %, furthermore preferably up to 15 mol %, morepreferably up to 10 mol %, even more preferably up to 5 mol %, likewisemore preferably up to 2 mol % of insoluble or sparingly soluble salts.Salts which are sparingly soluble in the context of the presentinvention are those of which the solubility product is <10⁻⁴. Saltswhich are readily soluble are those of which the solubility product is>10⁻⁴. Preferably, the injection buffer optionally contained in thepharmaceutical composition according to the invention is from 50 mM to800 mM, preferably from 60 mM to 500 mM, more preferably from 70 mM to250 mM, particularly preferably 60 mM to 110 mM in sodium chloride(NaCl), from 0.01 mM to 100 mM, preferably from 0.5 mM to 80 mM, morepreferably from 1.5 mM to 40 mM in calcium chloride (CaCl₂) andoptionally from 3 mM to 500 mM, preferably from 4 mM to 300 mM, morepreferably from 5 mM to 200 mM in potassium chloride (KCl). Organicanions can also occur as further anions in addition to theabovementioned inorganic anions, for example halides, sulfates orcarbonates. Among these there may be mentioned succinate, lactobionate,lactate, malate, maleate etc., which can also be present in combination.

An injection buffer optionally contained in the (pharmaceutical)composition according to the invention preferably contains lactate. Ifit contains an organic anion, such an injection buffer particularlypreferably contains exclusively lactate as the organic anion. Lactate inthe context of the invention can be any desired lactate, for exampleL-lactate and D-lactate. Lactate salts which occur in connection withthe present invention are typically sodium lactate and/or calciumlactate, especially if the injection buffer contains only Na⁺ as amonovalent cation and Ca²⁺ as a divalent cation. An injection bufferoptionally used in the (pharmaceutical) composition according to theinvention and as described above preferably contains, with respect tothe total pharmaceutical composition, from 15 mM to 500 mM, morepreferably from 15 mM to 200 mM, and even more most preferably from 15mM to 100 mM lactate.

If formulated in non-liquid form (e.g. in solid or semi-solid form), thepharmaceutical composition of the invention may be contain compoundswhich can serve as suitable carriers or constituents thereof, e.g.sugars, such as, for example, lactose, glucose and sucrose; starches,such as, for example, corn starch or potato starch; cellulose and itsderivatives, such as, for example, sodium carboxymethylcellulose,ethylcellulose, cellulose acetate; pulverized tragacanth; malt;gelatine; tallow; solid lubricants, such as, for example, stearic acid,magnesium stearate; calcium sulfate; plant oils, such as, for example,groundnut oil, cottonseed oil, sesame oil, olive oil, corn oil and oilfrom Theobroma; polyols, such as, for example, polypropylene glycol,glycerol, sorbitol, mannitol and polyethylene glycol; alginic acid.However, the above compounds may also be used for the provision ofliquid compositions.

Other components which may be included in a pharmaceutical compositionof the invention are e.g. emulsifiers, such as, for example, Tween®;wetting agents, such as, for example, sodium lauryl sulfate; colouringagents; flavouring agents; medicament carriers; tablet-forming agents;stabilizers; antioxidants; preservatives.

Other suitable carriers for injection include hydrogels, devices forcontrolled or delayed release, polylactic acid and collagen matrices.Suitable carriers which can be used here include those which aresuitable for use in lotions, creams, gels and the like. If the compoundis to be administered perorally, tablets, capsules and the like are thepreferred unit dose form. The suitable carriers for the preparation ofunit dose forms which can be used for oral administration are well-knownin the prior art. Their choice will depend on secondary considerations,such as flavour, cost and storage stability, which are not critical forthe purposes of the present invention and can be implemented withoutdifficulties by a person skilled in the art.

The present invention also provides an (in vitro or in vivo)transfection method for transfecting cells or a tissue with thecomplexed RNA of the present invention as described above. The inventive(in vitro or in vivo) transfection method preferably comprises thefollowing steps:

-   -   a) Optionally preparing and/or providing a complexed RNA        according to the present invention, comprising at least one RNA        complexed with one or more oligopeptides having the empirical        formula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x);    -   b) Transfecting a cell, a (living) tissue or an organism (in        vitro or in vivo) using the complexed RNA prepared and/or        provided according to step a).

Preparing and/or providing a complexed RNA as defined above according tostep a) of the inventive in vitro or in vivo transfection method fortransfecting cells or a tissue with the complexed RNA of the presentinvention, may be carried out by any method known in the art. Acomplexed RNA as used herein comprises at least one RNA complexed withone or more oligopeptides having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). Preparing and/orproviding a complexed RNA as defined above according to step a) may thuscomprise the preparation and/or provision of the least one RNA and theone or more oligopeptides having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x).

Methods for preparation of short peptide sequences such as(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), are widely known inthe art and may employ e.g. solid phase synthesis such as Fmoc solidphase synthesis or other suitable methods (see e.g. R. Martin, Ed.,Protein Synthesis: Methods and Protocols. Methods in Molecular Biology,Vol. 77 Humana Press (1998)).

Preparing and/or providing the at least one RNA (molecule) as componentof the inventive complex as defined above may comprise according to stepa) a first sub-step a1), namely provision and/or preparation of anucleic acid template, which typically comprises a sequencecorresponding to the desired RNA. The sequence of the nucleic acidtemplate may be any nucleic acid, e.g. a single- or double-stranded DNA,cDNA, genomic DNA or fragments thereof, etc., which may code for atherapeutically active protein, an antibody or an antigene, or any otherprotein or peptide as described above. Typically, DNA sequences, e.g.DNA plasmids, preferably in linearized form, may be employed for thispurpose. Preferably, the sequence of the nucleic acid template may be an(expression) vector, more preferably an (expression)vector having an RNApolymerase binding site. Any (expression) vectors known in the priorart, e.g. commercially available (expression) vectors (see above), canbe used for this. Preferred (expression) vectors are, for example, thosewhich have an SP6 or a T7 or T3 binding site upstream and/or downstreamof the cloning site. The vector may comprise a nucleic acid sequenceencoding a therapeutically active protein, an antibody or an antigen, orany other protein or peptide as described above, which is typicallycloned into the (expression) vector, e.g. via a multiple cloning site ofthe vector used.

Prior to transcription the (expression) vector is typically cleaved withrestriction enzymes at the site at which the future 3′ end of the RNA isto be found, using a suitable restriction enzyme, and the fragment ispurified. This prevents the transcribed RNA from containing vectorsequences, and an RNA transcript of defined length may be obtained. Inthis context, preferably no restriction enzymes which generateoverhanging ends (such as, e.g., AatII, ApaI, BanII, BglI, Bsp1286,BstXI, CfoI, HaeII, HgiAI, HhaI, KpnI, PstI, PvuI, SacI, SacII, SfiI,SphI, etc.) are used. Should such restriction enzymes nevertheless beused, the overhanging 3′ end preferably may be filled up, e.g. withKlenow or T4 DNA polymerase.

As an alternative to the above, the nucleic acid template used forpreparing and/or providing the at least one RNA (molecule) of thecomplexed RNA of the invention, may be prepared by employing apolymerase chain reaction (PCR). The nucleic acid template preferablyand one of the primers used therefore, typically contains the sequenceof an RNA polymerase binding site. Furthermore, the 5′ end of the primerused preferably contains an extension of about 10-50 furthernucleotides, more preferably of from 15 to 30 further nucleotides andmost preferably of about 20 nucleotides.

Prior to in vitro transcription, the nucleic acid, e.g. the DNA or cDNAtemplate, used as transcription template, is typically purified and freefrom RNase in order to ensure a high yield. In this context,purification of such template can be carried out with the aid of anymethod known in the prior art, for example using a caesium chloridegradient, ion exchange methods or by purification via agarose gelelectrophoresis.

Subsequent to preparing and/or providing the nucleic acid template, anin vitro transcription reaction according to a second sub-step a2) maybe carried out for preparing the desired the at least one RNA (molecule)of the complexed RNA of the invention using the nucleic acid templateprepared according to first sub-step a1) as defined above.

The in vitro transcription reaction according to a second sub-step a2)is typically carried out in an in vitro transcription reaction. Asuitable in vitro transcription medium initially comprises the nucleicacid template as described above, for example about 0.1-10 μg,preferably about 1-5 μg, more preferably 2.5 μg and most preferablyabout 1 μg of such a nucleic acid. A suitable in vitro transcriptionmedium furthermore optionally comprises a reducing agent, e.g. DTT, morepreferably about 1-20 μl 50 mM DTT, even more preferably about 5 μl 50mM DTT. The in vitro transcription medium typically comprisesnucleotides, e.g. a nucleotide mix, in the case of the present inventioncomprising a mixture of nucleotides of A, G, C or U, typically about0.1-10 mM per nucleotide, preferably 0.1 to 1 mM per nucleotide,preferably about 4 mM in total. A suitable in vitro transcription mediumlikewise comprises an RNA polymerase, e.g. T7 RNA polymerase (forexample T7-Opti mRNA Kit, CureVac, Tübingen, Germany), T3 RNA polymeraseor SP6, typically about 10 to 500 U, preferably about 25 to 250 U, morepreferably about 50 to 150 U, and most preferably about 100 U of RNApolymerase. The in vitro transcription medium is furthermore preferablykept free from RNase in order to avoid degradation of the transcribedthe at least one RNA (molecule) of the complexed RNA of the invention. Asuitable in vitro transcription medium therefore optionally additionallycomprises an RNase inhibitor.

The nucleic acid template may be then incubated in the in vitrotranscription medium and is transcribed to the at least one RNA(molecule) of the complexed RNA of the invention, which may encode for atherapeutically active protein, an antibody or an antigene, or any otherprotein or peptide as described above. The incubation times aretypically about 30 to 240 minutes, preferably about 40 to 120 minutesand most preferably about 90 minutes. The incubation temperatures aretypically about 30-45° C., preferably 37-42° C. The incubationtemperature depends on the RNA polymerase used, e.g. for T7 RNApolymerase it is about 37° C. The at least one RNA (molecule) of thecomplexed RNA of the invention obtained by the transcription ispreferably an mRNA. The yields obtained in the in vitro transcriptionare, for the stated starting amounts employed above, typically in theregion of about 30 μg of RNA per μg of template DNA used. In the contextof the present invention, the yields obtained in the in vitrotranscription can be increased by linear up scaling. For this, thestated starting amounts employed above are preferably increasedaccording to the yields required, e.g. by a multiplication factor of 5,10, 50, 100, 500, 1,000, 5,000, 10,000, 50,000, 100,000 etc.

After incubation, a purification of the transcribed at least one RNA(molecule) of the complexed RNA of the invention can optionally takeplace. Any suitable method known in the prior art, e.g. chromatographicpurification methods, e.g. affinity chromatography, gel filtration etc.,can be used for this. By the purification, non-incorporated, i.e. excessnucleotides and template DNA can be removed from the in vitrotranscription medium and a clean RNA can be obtained. For example, afterthe transcription the reaction mixture with the transcribed RNA cantypically be digested with DNase in order to remove the DNA templatestill contained in the reaction mixture. The transcribed at least oneRNA (molecule) of the complexed RNA of the invention can be subsequentlyor alternatively precipitated with LiCl. Purification of the transcribedRNA can then take place via IP RP-HPLC. This renders in particulareffective separation of longer and shorter fragments from one anotherpossible.

Preferably, in this context purification of the RNA may take place via amethod for purification of RNA on a preparative scale, which isdistinguished in that the RNA is purified by means of HPLC using aporous reverse phase as the stationary phase (PURE Messenger). Forexample, for the purification a reverse phase can be employed as thestationary phase for the HPLC purification. For the chromatography withreverse phases, a non-polar compound typically serves as stationaryphases, and a polar solvent, such as mixtures of water, which is usuallyemployed in the form of buffers, with acetonitrile and/or methanol,serves as the mobile phase for the elution. Preferably, the porousreverse phase has a particle size of 8.0±2 μm, preferably ±1 μm, morepreferably +/−0.5 μm. The reverse phase material can be in the form ofbeads. The purification can be carried out in a particularly favourablemanner with a porous reverse phase having this particle size, optionallyin the form of beads, particularly good separation results beingobtained. The reverse phase employed is preferably porous since withstationary reverse phases which are not porous, such as are describede.g. by Azarani A. and Hecker K. H., pressures which are too high arebuilt up, so that preparative purification of the RNA is possible, if atall, only with great difficulty. The reverse phase preferably has a poresize of from 200 to 5,000, in particular a pore size of from 300 to4,000. Particularly preferred pore sizes for the reverse phases are200-400, 800-1,200 and 3,500-4,500. With a reverse phase having thesepore sizes, particularly good results are achieved in respect of thepurification of the transcribed RNA. The material for the reverse phaseis preferably a polystyrene-divinylbenzene, and non-alkylatedpolystyrene-divinylbenzenes can be employed in particular. Stationaryphases with polystyrene-divinylbenzene are known per se. For thepurification, the polystyrene-divinylbenzenes which are known per se andalready employed for HPLC methods and are commercially obtainable can beused. A non-alkylated porous polystyrene-divinylbenzene which inparticular has a particle size of 8.0±0.5 μm and a pore size of 250-300,900-1,100 or 3,500-4,500 is very particularly preferably used for thepurification. The advantages described above can be achieved in aparticularly favourable manner with this material for the reversephases.

The HPLC purification can be carried out by the ion pair method, an ionhaving a positive charge being added to the mobile phase as acounter-ion to the negatively charged RNA. An ion pair having alipophilic character, which is slowed down by the non-polar stationaryphase of the reverse phase system, is formed in this manner. Inpractice, the precise conditions for the ion pair method must be workedout empirically for each specific separation problem. The size of thecounter-ion, its concentration and the pH of the solution contributegreatly towards the result of the separation. In a favourable manner,alkylammonium salts, such as triethylammonium acetate and/ortetraalkylammonium compounds, such as tetrabutylammonium, are added tothe mobile phase. Preferably, 0.1 M triethylammonium acetate is addedand the pH is adjusted to about 7. The choice of mobile phase depends onthe nature of the desired separation. This means that the mobile phasefound for a specific separation, such as can be known, for example, fromthe prior art, cannot be transferred readily to another separationproblem with adequate prospect of success. The ideal elution conditions,in particular the mobile phase used, must be determined for eachseparation problem by empirical experiments. A mixture of an aqueoussolvent and an organic solvent can be employed as the mobile phase forelution of the RNA by the HPLC method. In this context, it is favourableif a buffer which has, in particular, a pH of about 7, for example6.5-7.5, e.g. 7.0, is used as aqueous solvent; preferably, the buffertriethylammonium acetate is used, particularly preferably a 0.1 Mtriethylammonium acetate buffer which, as described above, also acts asa counter-ion to the RNA in the ion pair method. The organic solventemployed in the mobile phase can be acetonitrile, methanol or a mixtureof these two, very particularly preferably acetonitrile. Thepurification of the RNA using an HPLC method as described is carried outin a particularly favourable manner with these organic solvents. Themobile phase is particularly preferably a mixture of 0.1 Mtriethylammonium acetate, pH 7, and acetonitrile. It has emerged to belikewise particularly favourable if the mobile phase contains 5.0 vol. %to 20.0 vol. % of organic solvent, based on the mobile phase, and theremainder to make up 100 vol. % is the aqueous solvent. It is veryparticularly favourable for the method according to the invention if themobile phase contains 9.5 vol. % to 14.5 vol. % of organic solvent,based on the mobile phase, and the remainder to make up 100 vol. % isthe aqueous solvent. Elution of the RNA can subsequently be carried outisocratically or by means of a gradient separation. In the case of anisocratic separation, elution of the RNA is carried out with a singleeluting agent or a mixture of several eluting agents which remainsconstant, it being possible for the solvents described above in detailto be employed as the eluting agent.

Alternatively, the at least one RNA (molecule) according to step a) ofthe inventive method of transfection may well be prepared by chemicalsynthesis. Hereby, various methods known in the art may be used. Thephosphoroamidite method is used most widely as a method of chemicallysynthesizing oligonucleotides, e.g. RNA fragments (Nucleic AcidResearch, 17:7059-7071, 1989). In general, this phosphoroamidite methodmakes use of a condensation reaction between a nucleosidephosphoroamidite and a nucleoside as a key reaction using tetrazole asan accelerator. Because this reaction usually occurs competitively onboth the hydroxyl group in a sugar moiety and the amino group in anucleoside base moiety, the selective reaction on only the hydroxylgroup in a sugar moiety is required to synthesize a desired nucleotide.Accordingly, the side reaction on the amino group is usually preventedby protecting the amino group. The protective group is removed whensynthesis is finished. More specific information about how to synthesizeRNA molecules may be retrieved from Arnold et al., “Chloridite andAmidite Automated Synthesis of Oligodeoxyribonucleotides Using AmidineProtected Nucleosides,” reported in “7th Symposium Chem. Nucleic AcidComponents,” Nucleic Acids Symposium Series, 18, 181-184 (Aug. 30,1987); Chemical Abstracts, 108(19), p. 692, Abstr. No. 167875z (May 9,1988); Hayakawa et al., “Benzimidazolium Triflate as an EfficientPromoter for Nucleotide Synthesis via the Phosphoramidite Method,” J.Organic Chemistry, 61(23), 7996-7997 (Nov. 15, 1996); Pirrung et al.,“Proofing of Photolithographic DNA Synthesis with3′,5′-Dimethoxybenzoinyloxycarbonyl-Protected DeoxynucleosidePhosphoramidites,” J. Organic Chemistry, 63(2), 241-246 (Jan. 23, 1998);Effenberger et al., Trifluoromethanesulfonic Imidazolide—A ConvenientReagent for Introducing the Triflate Group, Tetrahedron Letters, 1980(45), 3947-3948 (September 1980), all of them incorporated herein byreference.

Preparation of the complexed RNA according to step a) of the presentinvention typically occurs according to sub-step a3) by adding aspecific amount of the at least one RNA (molecule) to a specific amountto the one or more oligopeptides having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x). Thereby, molar ormass ratios as indicated above of the at least one RNA (molecule) andthe one or more oligopeptides having the herein defined empiricalformula (Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) are typicallyenvisaged. Complex formation typically occurs upon mixing bothcomponents. Thereby, the peptidic component is typically added o the RNAcomponent, in some cases, however, vice versa.

Such a preparation step according to method step a), however, isoptional and may not take place if the complexed RNA according to thepresent invention is already available. Accordingly, sub-steps a1), a2)and a3) as defined above are also optional and need not to be carriedout, if the RNA used for the complexed RNA is already available.Similarly, the one or more oligopeptides having the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x) may be used directlyand need not to be prepared, if already available, e.g. from a supplier.

According to step b) of the inventive method for transfecting cells ortissues in vitro or in vivo a cell or a tissue may be transfected usingthe complexed RNA provided and/or prepared according to step a).Transfection of the cells or tissues in vitro or in vivo is in generalcarried out by adding the complexed RNA provided and/or preparedaccording to step a) to the cells or tissue. Preferably, the complexedRNA then enters into the cells by using cellular mechanisms, e.g.endocytosis. Addition of the complexed RNA as such to the cells ortissues may occur according to the invention without addition of anyfurther components due to the transfectional potential of the complexedRNA (molecule) of the invention. Alternatively, addition of thecomplexed RNA provided and/or prepared according to step a) to the cellsor tissue may occur in the form of a composition, e.g. as component ofan aqueous solution, preferably a pharmaceutical composition as definedabove, which may optionally contain additional components for furtherenhancement of the transfection activity.

Cells (or host cells) in this context for transfection of the complexedRNA (provided and/or prepared according to step a)) in vitro includesany cell, and preferably, with out being restricted thereto, cells,which shall be transfected by any RNA molecule (as defined above) byusing the inventive complexed RNA. In particular, RNA transfection mayallow for expression of a protein encoded by the RNA of the complexedRNA according to the invention in the cell or may allow RNA (e.g. siRNA,anti-sense RNA) of the inventive complex to attenuate or suppress theexpression of a cellular gene. Cells in this context preferably includecultured eukaryotic cells (e.g. yeast cells, plant cells, animal cellsand human cells) or prokaryotic cells (e.g. bacteria cells etc.) orinduce an immune response. Cells of multicellular organisms arepreferably chosen if posttranslational modifications, e.g. glycosylationof the encoded protein, are necessary (N- and/or O-coupled). In contrastto prokaryotic cells, such (higher) eukaryotic cells renderposttranslational modifications of the protein synthesized possible. Theperson skilled in the art knows a large number of such higher eukaryoticcells or cell lines, e.g. 293T (embryonal kidney cell line), HeLa (humancervix carcinoma cells), CHO (cells from the ovaries of the Chinesehamster) and further cell lines, including such cells and cell linesdeveloped for laboratory purposes, such as, for example, hTERT-MSC,HEK293, Sf9 or COS cells. Suitable eukaryotic cells furthermore includecells or cell lines which are impaired by diseases or infections, e.g.cancer cells, in particular cancer cells of any of the types of cancermentioned here in the description, cells impaired by HIV, and/or cellsof the immune system or of the central nervous system (CNS). Suitablecells can likewise be derived from eukaryotic microorganisms, such asyeast, e.g. Saccharomyces cerevisiae (Stinchcomb et al., Nature, 282:39,(1997)), Schizosaccharomyces pombe, Candida, Pichia, and filamentousfungi of the genera Aspergillus, Penicillium, etc. Suitable cellslikewise include prokaryotic cells, such as e.g. bacteria cells, e.g.from Escherichia coli or from bacteria of the general Bacillus,Lactococcus, Lactobacillus, Pseudomonas, Streptomyces, Streptococcus,Staphylococcus, preferably E. coli, etc. Human cells or animal cells,e.g. of animals as mentioned herein, are particularly preferred aseukaryotic cells. Furthermore, antigen presenting cells (APCs) may beused for ex vivo transfection of the complexed RNA according to thepresent invention. Particularly preferred are dendritic cells, which maybe used for ex vivo transfection of the complexed RNA according to thepresent invention.

According to a particularly preferred embodiment, blood cells and/orhaemopoietic cells, or partial populations thereof, i.e. any type ofcells, which may be isolated from (whole) blood and/or which may bederived from cultivated cell lines derived from those cells, may betransfected with a complexed RNA as defined herein using the abovemethod of transfection, e.g. red blood cells (erythrocytes),granulocytes, mononuclear cells (peripheral blood mononuclear cells,PBMCs) and/or blood platelets (thrombocytes), APSs, DCs, etc.Preferably, blood cells are used, especially partial populationsthereof, which are characterized in particular in that they contain asmall proportion of well-differentiated professional APCs, such as DCs.The transfected cells may contain preferably less than 5%, particularlypreferably no more than 2%, of DCs when used for transfection. In thecontext of the present invention “blood cells” are preferably understoodas a mixture or an enriched to substantially pure population of redblood cells, granulocytes, mononuclear cells (PBMCs) and/or bloodplatelets from whole blood, blood serum or another source, e.g. from thespleen or lymph nodes, only a small proportion of professional APCsbeing present. The blood cells as used according to the presentinvention are preferably fresh blood cells, i.e. the period betweencollection of the blood cells (especially blood withdrawal) andtransfection being only short, e.g. less than 12 h, preferably less than6 h, particularly preferably less than 2 h and very particularlypreferably less than 1 h. Furthermore, the blood cells to be transfectedusing the above method for transfecting the complexed RNA according tothe present invention preferably originate from the actual patient whowill be treated with the pharmaceutical composition of the presentinvention. The use of blood cells, haematopoietic cells or partialpopulations thereof as defined above is based on the surprisingdiscovery that for vaccination of a patient to be treated againstcertain antigens encoded by an mRNA as defined herein, it is notnecessary to differentiate blood cells, e.g. PBMCs, obtained e.g. fromthe blood of an individual, especially the actual patient to be treated,by means of laborious, lengthy and expensive cell culture techniques,into a population of cells with a high proportion of professionalantigen presenting cells (APCs), especially dendritic cells (DCs), butthat it is sufficient, for a successful immune stimulation, to transfectblood cells directly with the mRNA coding for one or more antigens inorder to obtain a pharmaceutical composition which effects a suitableimmune stimulation e.g. in the actual patient from whom the blood cells,especially the abovementioned partial populations thereof, have beenobtained, said immune stimulation preferably being directed against oneor more antigens from a tumour or one or more antigens from a pathogenicgerm or agent. Transfection of a complexed RNA as defined herein intoblood cells or cells derived therefrom (either isolated therefrom orfrom respective cultivated cell lines) is not limited to antigens and,of course, relates to any RNA as defined herein used for a complexedRNA, e.g. any further immunostimulating RNA as defined herein, anycoding RNA, etc.

While there is the need to transfect cultivated cells in vitro (e.g.human or animal cells) or to transfect explanted cells (e.g. human oranimal cells) in vitro (before retransplantation into the hostorganism), direct administration of the complexed RNA of the inventionto patients for in vivo transfection is envisaged as well. Accordingly,transfection of the complexed RNA (provided and/or prepared according tostep a)) may also occur in vivo according to step b), i.e. may beadministered to living tissues and/or organisms. Therefore, thecomplexed RNA provided according to step a) of the inventivetransfection method may be administered to a living tissue or anorganism either as such or e.g. as component of a (liquid) composition,in particular an aqueous composition, e.g. a pharmaceutical compositionas defined above. In this context, an organism (or a being) typicallymeans mammals, selected from, without being restricted thereto, thegroup comprising humans, and animals, including e.g. pig, goat, cattle,swine, dog, cat, donkey, monkey, ape or rodents, including mouse,hamster and rabbit. Furthermore, living tissues as mentioned above, arepreferably derived from these organisms. Administration of the complexedRNA to those living tissues and/or organisms may occur via any suitableadministration route, e.g. systemically, and include e.g. intra- ortransdermal, oral, parenteral, including subcutaneous, intramuscular orintravenous injections, topical and/or intranasal routes as definedabove.

Moreover, the method for transfection, which may be used in vitro or exvivo, may also be well suited for use in vivo, e.g. as method oftreatment of various diseases as mentioned herein. In a preferred formof a method of treatment according to the invention a further step maybe included, which may contain administration of anotherpharmaceutically effective substance, e.g. an antibody, an antigen (inparticular a pathogenic or a tumor antigen as disclosed herein) or theadministration of at least one cytokine. Both may be administeredseparately from the complexed RNA as DNA or RNA coding for e.g. thecytokine or the antigen or the cytokine or antigen may be administeredas such. The method of treatment may also comprise the administration ofan additional adjuvant (as disclosed herein), which may further activatethe immune system.

According to a further embodiment of the present invention, thecomplexed RNA as defined above, comprising at least one RNA complexedwith one or more oligopeptides, wherein the oligopeptide shows a lengthof 8 to 15 amino acids and has the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), may be used fortreatment and/or prophylaxis of specific diseases as mentioned herein.Treatment and/or prophylaxis of specific diseases is typically dependenton selection of a suitable protein encoded by the RNA of the complexedRNA of the present invention. Treatment in this context may occur eitherby administering the complexed RNA according to the present invention(encoding this protein) as such or by administering the (pharmaceutical)composition according to the present invention as defined above.

Without being limited thereto, diseases or states include in thiscontext, for example, cancer or tumour diseases chosen from melanomas,malignant melanomas, colon carcinomas, lymphomas, sarcomas, blastomas,kidney carcinomas, gastrointestinal tumours, gliomas, prostate tumours,bladder cancer, rectal tumours, stomach cancer, oesophageal cancer,pancreatic cancer, liver cancer, mammary carcinomas (=breast cancer),uterine cancer, cervical cancer, acute myeloid leukaemia (AML), acutelymphoid leukaemia (ALL), chronic myeloid leukaemia (CML), chroniclymphocytic leukaemia (CLL), hepatomas, diverse virus-induced tumours,such as e.g. papilloma virus-induced carcinomas (e.g. cervixcarcinoma=cervical cancer), adenocarcinomas, herpes virus-inducedtumours (e.g. Burkitt's lymphoma, EBV-induced B cell lymphoma),hepatitis B-induced tumours (hepatocell carcinomas), HTLV-1- andHTLV-2-induced lymphomas, acusticus neurinoma, lung carcinomas (=lungcancer=bronchial carcinoma), small cell lung carcinomas, throat cancer,anal carcinoma, glioblastoma, rectum carcinoma, astrocytoma, braintumours, retinoblastoma, basalioma, brain metastases, medulloblastomas,vaginal cancer, testicular cancer, thyroid carcinoma, Hodgkin'ssyndrome, meningeomas, Schneeberger's disease, pituitary tumour, mycosisfungoides, carcinoids, neurinoma, spinalioma, Burkitt's lymphoma,laryngeal cancer, kidney cancer, thymoma, corpus carcinoma, bone cancer,non-Hodgkin's lymphomas, urethral cancer, CUP syndrome, head/necktumours, oligodendroglioma, vulval cancer, intestinal cancer, coloncarcinoma, oesophageal carcinoma (=oesophageal cancer), wart conditions,small intestine tumours, craniopharyngeomas, ovarian carcinoma, softtissue tumours, ovarian cancer (=ovarian carcinoma), pancreaticcarcinoma (=pancreatic cancer), endometrium carcinoma, liver metastases,penis cancer, tongue cancer, gallbladder cancer, leukaemia,plasmocytoma, lid tumour, prostate cancer (=prostate tumours) etc.

Diseases or states may also include in this context infectious diseaseschosen from, e.g., viral infectious diseases chosen from, without beinglimited thereto, SARS, yellow fever, Lyme, anthrax, AIDS, condylomaacuminata, molluscum contagiosum, dengue fever, three-day fever, Ebolavirus, colds, early summer meningoencephalitis (ESME), influenza,shingles, hepatitis, herpes simplex type I, herpes simplex type II,herpes zoster, influenza, Japanese encephalitis, Lassa fever, Marburgvirus, measles, foot and mouth disease, mononucleosis, mumps, Norwalkvirus infection, Pfeiffer's glandular fever, smallpox, polio(poliomyelitis), pseuodcroup, infectious erythema, rabies, warts, WestNile fever, chicken-pox, cytomegalovirus (CMV), bacterial infectiousdiseases, such as abortion (prostate inflammation), anthrax,appendicitis (inflammation of the caecum), borreliosis, botulism,Campylobacter, Chlamydia trachomatis (inflammation of the urethra,conjunctiva), cholera, diphtheria, donavonosis, epiglottitis,louse-borne typhus, typhoid fever, gas gangrene, gonorrhoea, hareplague, Helicobacter pylori, whooping-cough, climatic bubo,osteomyelitis, legionnaires' disease, leprosy, listeriosis, pneumonia,meningitis, bacterial meningitis, anthrax, inflammation of the middleear, Mycoplasma hominis, neonatal sepsis (chorioamnionitis), noma,paratyphoid fever, plague, Reiter's syndrome, Rocky Mountain spottedfever, Salmonella paratyphoid fever, Salmonella typhoid fever, scarletfever, syphilis, tetanus, gonorrhoea, tsutsugamushi fever, tuberculosis,typhus, vaginitis (colpitis), soft chancre and infectious diseasescaused by parasites, protozoa or fungi, such as amoebic dysentery,bilharziosis, Chagas' disease, Echinococcus, fish tapeworm,ichthyotoxism (ciguatera), fox tapeworm, mycosis pedis, dog tapeworm,candiosis, ptyriasis, the itch (scabies), leishmaniasis, cutaneousleishmaniasis, lamblian dysentery (giadiasis), lice, malaria,microscopy, onchocercosis (river blindness), fungal diseases, beeftapeworm, schistosomiasis, sleeping sickness, pork tapeworm,toxoplasmosis, trichomoniasis, trypanosomiasis (sleeping sickness),visceral leishmaniasis, nappy dermatitis or dwarf tapeworm.

Diseases in the context of the present invention likewise include,without being limited thereto, (infectious) virus diseases caused byviruses chosen from, without being limited thereto, HIV, orthopoxvariola virus, orthopox alastrim virus, parapox ovis virus, molluscumcontagiosum virus, herpes simplex virus 1, herpes simplex virus 2,herpes B virus, varicella zoster virus, pseudorabies virus, humancytomegaly virus, human herpes virus 6, human herpes virus 7,Epstein-Barr virus, human herpes virus 8, hepatitis B virus, chikungunyavirus, O'nyong'nyong virus, rubivirus, hepatitis C virus, GB virus C,West Nile virus, dengue virus, yellow fever virus, louping ill virus,St. Louis encephalitis virus, Japan B encephalitis virus, Powassanvirus, FSME virus, SARS-associated corona virus, human corona virus229E, human corona virus Oc43, Torovirus, human T cell lymphotropicvirus type I, human T cell lymphotropic virus type II, humanimmunodeficiency virus type 1, human immunodeficiency virus type 2,Lassa virus, lymphocytic choriomeningitis virus, Tacaribe virus, Juninvirus, Machupo virus, Borna disease virus, Bunyamwera virus, Californiaencephalitis virus, Rift Valley fever virus, sand fly fever virus,Toscana virus, Crimean-Congo haemorrhagic fever virus, Hazara virus,Khasan virus, Hantaan virus, Seoul virus, Prospect Hill virus, Puumalavirus, Dobrava Belgrade virus, Tula virus, sin nombre virus, LakeVictoria Marburg virus, Zaire Ebola virus, Sudan Ebola virus, IvoryCoast Ebola virus, influenza virus A, influenza virus B, influenzaviruses C, parainfluenza virus, measles virus, mumps virus, respiratorysyncytial virus, human metapneumovirus, vesicular stomatitis Indianavirus, rabies virus, Mokola virus, Duvenhage virus, European batlyssavirus 1+2, Australian bat lyssavirus, adenoviruses A-F, humanpapilloma viruses, condyloma virus 6, condyloma virus 11, polyomaviruses, adeno-associated virus 2, rotaviruses, or orbiviruses etc.These diseases may e.g. be treated by a vaccine according to theinvention.

Additionally, diseases or states may include cardiovascular diseaseschosen from, without being limited thereto, coronary heart disease,arteriosclerosis, apoplexy and hypertension, and neuronal diseaseschosen from Alzheimer's disease, amyotrophic lateral sclerosis,dystonia, epilepsy, multiple sclerosis and Parkinson's disease etc.

Diseases or states may also include in this context an allergic disorderor disease. Allergy is a condition that typically involves an abnormal,acquired immunological hypersensitivity to certain foreign antigens orallergens. Allergies normally result in a local or systemic inflammatoryresponse to these antigens or allergens and leading to an immunity inthe body against these allergens. Allergens in this context include e.g.grasses, pollens, molds, drugs, or numerous environmental triggers, etc.Without being bound to any theory, several different disease mechanismsare supposed to be involved in the development of allergies. Accordingto a classification scheme by P. Gell and R. Coombs the word “allergy”was restricted to type I hypersensitivities, which are caused by theclassical IgE mechanism. Type I hypersensitivity is characterised byexcessive activation of mast cells and basophils by IgE, resulting in asystemic inflammatory response that can result in symptoms as benign asa runny nose, to life-threatening anaphylactic shock and death. Wellknown types of allergies include, without being limited thereto,allergic asthma (leading to swelling of the nasal mucosa), allergicconjunctivitis (leading to redness and itching of the conjunctiva),allergic rhinitis (“hay fever”), anaphylaxis, angiodema, atopicdermatitis (eczema), urticaria (hives), eosinophilia, respiratory,allergies to insect stings, skin allergies (leading to and includingvarious rashes, such as eczema, hives (urticaria) and (contact)dermatitis), food allergies, allergies to medicine, etc. With regard tothe present invention, e.g. a pharmaceutical composition is provided,which contains e.g. an RNA coding for an allergen (e.g. from a catallergen, a dust allergen, a mite antigen, a plant antigen (e.g. a birchantigen) etc.) as a complex of the invention. Hereby, the encodedallergen may desensitize the patient' immune response. Alternatively,the pharmaceutical compositions of the present invention may shift the(exceeding) immune response to a stronger TH1 response, therebysuppressing or attenuating the undesired IgE response from the patientsuffers.

Furthermore, diseases or states as defined herein may include autoimmunediseases. Autoimmune diseases can be broadly divided into systemic andorgan-specific or localised autoimmune disorders, depending on theprincipal clinico-pathologic features of each disease. Autoimmunedisease may be divided into the categories of systemic syndromes,including SLE, Sjörgen's syndrome, Scleroderma, rheumatoid arthritis andpolyomyositis or local syndromes which may be endocrinologic (DM Type 1,Hashimoto's thyroiditis, Addison's disease, etc.), dermatologic(pemphigus vulgaris), haematologic (autoimmune haemolytic anaemia),neural (multiple sclerosis) or can involve virtually any circumscribedmass of body tissue. The autoimmune diseases to be treated may beselected from the group consisting of type I autoimmune diseases or typeII autoimmune diseases or type III autoimmune diseases or type IVautoimmune diseases, such as, for example, multiple sclerosis (MS),rheumatoid arthritis, diabetes, type I diabetes (Diabetes mellitus),systemic lupus erythematosus (SLE), chronic polyarthritis, Basedow'sdisease, autoimmune forms of chronic hepatitis, colitis ulcerosa,allergy type I diseases, allergy type II diseases, allergy type IIIdiseases, allergy type IV diseases, fibromyalgia, hair loss, Bechterew'sdisease, Crohn's disease, Myasthenia gravis, neurodermitis, Polymyalgiarheumatica, progressive systemic sclerosis (PSS), psoriasis, Reiter'ssyndrome, rheumatic arthritis, psoriasis, vasculitis, etc, or type IIdiabetes.

While the exact mode as to why the immune system induces a immunereaction against autoantigens has not been elucidated so far, there areseveral findings with regard to the etiology. Accordingly, theautoreaction may be due to a T-Cell Bypass. A normal immune systemrequires the activation of B-cells by T-cells before the former canproduce antibodies in large quantities. This requirement of a T-cell canbe by-passed in rare instances, such as infection by organisms producingsuper-antigens, which are capable of initiating polyclonal activation ofB-cells, or even of T-cells, by directly binding to the -subunit ofT-cell receptors in a non-specific fashion. Another explanation deducesautoimmune diseases from a molecular mimicry. An exogenous antigen mayshare structural similarities with certain host antigens; thus, anyantibody produced against this antigen (which mimics the self-antigens)can also, in theory, bind to the host antigens and amplify the immuneresponse. The most striking form of molecular mimicry is observed inGroup A beta-haemolytic streptococci, which shares antigens with humanmyocardium, and is responsible for the cardiac manifestations ofrheumatic fever. The present invention allows therefore to provide anRNA coding for an autoantigen as component of the complexed RNA of theinvention (or a (liquid) composition containing such a complexed RNA ofthe invention) or to provide a pharmaceutical composition containing anautoantigen (as protein, mRNA or DNA encoding for a autoantigen protein)and a complexed RNA of the invention all of which typically allow theimmune system to be desensitized.

Finally, diseases to be treated in the context of the present inventionlikewise include monogenetic diseases, i.e. (hereditary) diseases, orgenetic diseases in general. Such genetic diseases are typically causedby genetic defects, e.g. due to gene mutations resulting in loss ofprotein activity or regulatory mutations which do not allowtranscription or translation of the protein. Frequently, these diseaseslead to metabolic disorders or other symptoms, e.g. muscle dystrophy.Accordingly, the present invention allows to treat these diseases byproviding the complexed RNA as defined herein. Insofar, the followingdiseases may be treated: 3-beta-hydroxysteroid dehydrogenase deficiency(type II); 3-ketothiolase deficiency; 6-mercaptopurine sensitivity;Aarskog-Scott syndrome; Abetalipoproteinemia; Acatalasemia;Achondrogenesis; Achondrogenesis-hypochondrogenesis; Achondroplasia;Achromatopsia; Acromesomelic dysplasia (Hunter-Thompson type); ACTHdeficiency; Acyl-CoA dehydrogenase deficiency (short-chain, mediumchain, long chain); Adenomatous polyposis coli; Adenosin-deaminasedeficiency; Adenylosuccinase deficiency; Adhalinopathy; Adrenalhyperplasia, congenital (due to 11-beta-hydroxylase deficiency; due to17-alpha-hydroxylase deficiency; due to 21-hydroxylase deficiency);Adrenal hypoplasia, congenital, with hypogonadotropic hypogonadism;Adrenogenital syndrom; Adrenoleukodystrophy; Adrenomyeloneuropathy;Afibrinogenemia; Agammaglobulinemia; Alagille syndrome; Albinism (brown,ocular, oculocutaneous, rufous); Alcohol intolerance, acute; Aldolase Adeficiency; Aldosteronism, glucocorticoid-remediable; Alexander disease;Alkaptonuria; Alopecia universalis; Alpha-1-antichymotrypsi ndeficiency; Alpha-methylacyl-CoA racemase deficiency;Alpha-thalassemia/mental retardation syndrome; Alport syndrome;Alzheimer disease-1 (APP-related); Alzheimer disease-3; Alzheimerdisease-4; Amelogenesis imperfecta; Amyloid neuropathy (familial,several allelic types); Amyloidosis (Dutch type; Finnish type;hereditary renal; renal; senile systemic); Amytrophic lateral sclerosis;Analbuminemia; Androgen insensitivity; Anemia (Diamond-Blackfan); Anemia(hemolytic, due to PK deficiency); Anemia (hemolytic, Rh-null,suppressor type); Anemia (neonatal hemolytic, fatal and nearfatal);Anemia (sideroblastic, with ataxia); Anemia (sideroblastic/hypochromic);Anemia due to G6PD deficiency; Aneurysm (familial arterial); Angelmansyndrome; Angioedema; Aniridia; Anterior segment anomalies and cataract;Anterior segment mesenchymal dysgenesis; Anterior segment mesenchymaldysgenesis and cataract; Antithrombin III deficiency; Anxiety-relatedpersonality traits; Apert syndrome; Apnea (postanesthetic); ApoA-I andapoC-II deficiency (combined); Apolipoprotein A-II deficiency;Apolipoprotein B-100 (ligand-defective); Apparent mineralocorticoidexcess (hypertension due to); Argininemia; Argininosuccinicaciduria;Arthropathy (progressive pseudorheumatoid, of childhood);Aspartylglucosaminuria; Ataxia (episodic); Ataxia with isolated vitaminE deficiency; Ataxia-telangiectasia; Atelosteogenesis II; ATP-dependentDNA ligase I deficiency; Atrial septal defect with atrioventricularconduction defects; Atrichia with papular lesions; Autism(succinylpurinemic); Autoimmune polyglandular disease, type I; Autonomicnervous system dysfunction; Axenfeld anomaly; Azoospermia;Bamforth-Lazarus syndrome; Bannayan-Zonana syndrome; Barthsyndrome;Bartter syndrome (type 2 or type 3); Basal cell carcinoma; Basal cellnevus syndrome; BCG infection; Beare-Stevenson cutis gyrata syndrome;Becker muscular dystrophy; Beckwith-Wiedemann syndrome; Bernard-Souliersyndrome (type B; type C); Bethlem myopathy; Bile acid malabsorption,primary; Biotinidase deficiency; Bladder cancer; Bleeding disorder dueto defective thromboxane A2 receptor; Bloom syndrome; Brachydactyly(type B1 or type C); Branchiootic syndrome; Branchiootorenal syndrome;Breast cancer (invasive intraductal; lobular; male, with Reifensteinsyndrome; sporadic); Breast cancer-1 (early onset); Breast cancer-2(early onset); Brody myopathy; Brugada syndrome; Brunner syndrome;Burkitt lymphoma; Butterfly dystrophy (retinal); C1q deficiency (type A;type B; type C); C1r/C1s deficiency; C1s deficiency, isolated; C2deficiency; C3 deficiency; C3b inactivator deficiency; C4 deficiency; C8deficiency, type II; C9 deficiency; Campomelic dysplasia with autosomalsex reversal; Camptodactyly-arthropathy-coxa varapericarditis syndrome;Canavan disease; Carbamoylphosphate synthetase I deficiency;Carbohydrate-deficient glycoprotein syndrome (type I; type Ib; type II);Carcinoid tumor of lung; Cardioencephalomyopathy (fatal infantile, dueto cytochrome c oxidase deficiency); Cardiomyopathy (dilated; X-linkeddilated; familial hypertrophic; hypertrophic); Carnitine deficiency(systemic primary); Carnitine-acylcarnitine translocase deficiency;Carpal tunnel syndrome (familial); Cataract (cerulean; congenital;crystalline aculeiform; juvenile-onset; polymorphic and lamellar;punctate; zonular pulverulent); Cataract, Coppock-like; CD59 deficiency;Central core disease; Cerebellar ataxia; Cerebral amyloid angiopathy;Cerebral arteriopathy with subcortical infarcts and leukoencephalopathy;Cerebral cavernous malformations-1; Cerebrooculofacioskeletal syndrome;Cerebrotendinous xanthomatosis; Cerebrovascular disease; Ceroidlipofuscinosis (neuronal, variant juvenile type, with granularosmiophilic deposits); Ceroid lipofuscinosis (neuronal-1, infantile);Ceroid-lipofuscinosis (neuronal-3, juvenile); Char syndrome;Charcot-Marie-Tooth disease; Charcot-Marie-Tooth neuropathy;Charlevoix-Saguenay type; Chediak-Higashi syndrome; Chloride diarrhea(Finnish type); Cholestasis (benign recurrent intrahepatic); Cholestasis(familial intrahepatic); Cholestasis (progressive familialintrahepatic); Cholesteryl ester storage disease; Chondrodysplasiapunctata (brachytelephalangic; rhizomelic; X-linked dominant; X-linkedrecessive; Grebe type); Chondrosarcoma; Choroideremia; Chronicgranulomatous disease (autosomal, due to deficiency of CYBA); Chronicgranulomatous disease (X-linked); Chronic granulomatous disease due todeficiency of NCF-1; Chronic granulomatous disease due to deficiency ofNCF-2; Chylomicronemia syndrome, familial; Citrullinemia; classicalCockayne syndrome-1; Cleft lip, cleft jaw, cleft palate; Cleftlip/palate ectodermal dysplasia syndrome; Cleidocranial dysplasia; CMOII deficiency; Coats disease; Cockayne syndrome-2, type B; Coffin-Lowrysyndrome; Colchicine resistance; Colon adenocarcinoma; Colon cancer;Colorblindness (deutan; protan; tritan); Colorectal cancer; Combinedfactor V and VIII deficiency; Combined hyperlipemia (familial); Combinedimmunodeficiency (X-linked, moderate); Complex I deficiency; Complexneurologic disorder; Cone dystrophy-3; Cone-rod dystrophy 3; Cone-roddystrophy 6; Cone-rod retinal dystrophy-2; Congenital bilateral absenceof vas deferens; Conjunctivitis, ligneous; Contractural arachnodactyly;Coproporphyria; Cornea plana congenita; Corneal clouding; Cornealdystrophy (Avellino type; gelatinous drop-like; Groenouw type I; latticetype I; Reis-Bucklers type); Cortisol resistance; Coumarin resistance;Cowden disease; CPT deficiency, hepatic (type I; type II); Cramps(familial, potassium-aggravated); Craniofacial-deafness-hand syndrome;Craniosynostosis (type 2); Cretinism; Creutzfeldt-Jakob disease;Crigler-Najjar syndrome; Crouzon syndrome; Currarino syndrome; Cutislaxa; Cyclic hematopoiesis; Cyclic ichthyosis; Cylindromatosis; Cysticfibrosis; Cystinosis (nephropathic); Cystinuria (type II; type III);Daltonism; Darier disease; D-bifunctional protein deficiency; Deafness,autosomal dominant 1; Deafness, autosomal dominant 11; Deafness,autosomal dominant 12; Deafness, autosomal dominant 15; Deafness,autosomal dominant 2; Deafness, autosomal dominant 3; Deafness,autosomal dominant 5; Deafness, autosomal dominant 8; Deafness,autosomal dominant 9; Deafness, autosomal recessive 1; Deafness,autosomal recessive 2; Deafness, autosomal recessive 21; Deafness,autosomal recessive 3; Deafness, autosomal recessive 4; Deafness,autosomal recessive 9; Deafness, nonsyndromic sensorineural 13;Deafness, X-linked 1; Deafness, X-linked 3; Debrisoquine sensitivity;Dejerine-Sottas disease; Dementia (familial Danish); Dementia(frontotemporal, with parkinsonism); Dent disease; Dental anomalies;Dentatorubro-pallidoluysian atrophy; Denys-Drash syndrome;Dermatofibrosarcoma protuberans; Desmoid disease; Diabetes insipidus(nephrogenic); Diabetes insipidus (neurohypophyseal); Diabetes mellitus(insulin-resistant); Diabetes mellitus (rare form); Diabetes mellitus(type II); Diastrophic dysplasia; Dihydropyrimidinuria; Dosage-sensitivesex reversal; Doyne honeycomb degeneration of retina; Dubin-Johnsonsyndrome; Duchenne muscular dystrophy; Dyserythropoietic anemia withthrombocytopenia; Dysfibrinogenemia (alpha type; beta type; gamma type);Dyskeratosis congenita-1; Dysprothrombinemia; Dystonia (DOPAresponsive);Dystonia (myoclonic); Dystonia-1 (torsion); Ectodermal dysplasia;Ectopia lentis; Ectopia pupillae; Ectrodactyly (ectodermal dysplasia,and cleft lip/palate syndrome 3); Ehlers-Danlos syndrome (progeroidform); Ehlers-Danlos syndrome (type I; type II; type III; type IV; typeVI; type VII); Elastin Supravalvar aortic stenosis; Elliptocytosis-1;Elliptocytosis-2; Elliptocytosis-3; Ellis-van Creveld syndrome;Emery-Dreifuss muscular dystrophy; Emphysema; Encephalopathy;Endocardial fibroelastosis-2; Endometrial carcinoma; Endplateacetylcholinesterase deficiency; Enhanced S-cone syndrome; Enlargedvestibular aqueduct; Epidermolysis bullosa; Epidermolysis bullosadystrophica (dominant or recessive); Epidermolysis bullosa simplex;Epidermolytic hyperkeratosis; Epidermolytic palmoplantar keratoderma;Epilepsy (generalize; juvenile; myoclonic; nocturnal frontal lobe;progressive myoclonic); Epilepsy, benign, neonatal (type1 or type2);Epiphyseal dysplasia (multiple); Episodic ataxia (type 2); Episodicataxia/myokymia syndrome; Erythremias (alpha-; dysplasia);Erythrocytosis; Erythrokeratoderma; Estrogen resistance; Exertionalmyoglobinuria due to deficiency of LDH-A; Exostoses, multiple (type 1;type 2); Exudative vitreoretinopathy, X-linked; Fabry disease; Factor Hdeficiency; Factor VII deficiency; Factor X deficiency; Factor XIdeficiency; Factor XII deficiency; Factor XIIIA deficiency; Factor XIIIBdeficiency; Familial Mediterranean fever; Fanconi anemia; Fanconi-Bickelsyndrome; Farber lipogranulomatosis; Fatty liver (acute); Favism;Fish-eye disease; Foveal hypoplasia; Fragile X syndrome; Frasiersyndrome; Friedreich ataxia; fructose-bisphosphatase Fructoseintolerance; Fucosidosis; Fumarase deficiency; Fundus albipunctatus;Fundus flavimaculatus; G6PD deficiency; GABA-transaminase deficiency;Galactokinase deficiency with cataracts; Galactose epimerase deficiency;Galactosemia; Galactosialidosis; GAMT deficiency; Gardner syndrome;Gastric cancer; Gaucher disease; Generalized epilepsy with febrileseizures plus; Germ cell tumors; Gerstmann-Straussler disease; Giantcell hepatitis (neonatal); Giant platelet disorder; Giant-cellfibroblastoma; Gitelman syndrome; Glanzmann thrombasthenia (type A; typeB); Glaucoma 1A; Glaucoma 3A; Glioblastoma multiforme;Glomerulosclerosis (focal segmental); Glucose transport defect(blood-brain barrier); Glucose/galactose malabsorption; Glucosidase Ideficiency; Glutaricaciduria (type I; type IIB; type IIC); Gluthationsynthetase deficiency; Glycerol kinase deficiency; Glycine receptor(alpha-1 polypeptide); Glycogen storage disease I; Glycogen storagedisease II; Glycogen storage disease III; Glycogen storage disease IV;Glycogen storage disease VI; Glycogen storage disease VII; Glycogenosis(hepatic, autosomal); Glycogenosis (X-linked hepatic);GM1-gangliosidosis; GM2-gangliosidosis; Goiter (adolescentmultinodular); Goiter (congenital); Goiter (nonendemic, simple); Gonadaldysgenesis (XY type); Granulomatosis, septic; Graves disease; Greigcephalopolysyndactyly syndrome; Griscelli syndrome; Growth hormonedeficient dwarfism; Growth retardation with deafness and mentalretardation; Gynecomastia (familial, due to increased aromataseactivity); Gyrate atrophy of choroid and retina with ornithinemia (B6responsive or unresponsive); Hailey-Hailey disease; Haim-Munk syndrome;Hand-foot-uterus syndrome; Harderoporphyrinuria; HDL deficiency(familial); Heart block (nonprogressive or progressive); Heinz bodyanemia; HELLP syndrome; Hematuria (familial benign); Heme oxygenase-1deficiency; Hemiplegic migraine; Hemochromotosis; Hemoglobin H disease;Hemolytic anemia due to ADA excess; Hemolytic anemia due to adenylatekinase deficiency; Hemolytic anemia due to band 3 defect; Hemolyticanemia due to glucosephosphate isomerase deficiency; Hemolytic anemiadue to glutathione synthetase deficiency; Hemolytic anemia due tohexokinase deficiency; Hemolytic anemia due to PGK deficiency;Hemolytic-uremic syndrome; Hemophagocytic lymphohistiocytosis;Hemophilia A; Hemophilia B; Hemorrhagic diathesis due to factor Vdeficiency; Hemosiderosis (systemic, due to aceruloplasminemia); Hepaticlipase deficiency; Hepatoblastoma; Hepatocellular carcinoma; Hereditaryhemorrhagic telangiectasia-1; Hereditary hemorrhagic telangiectasia-2;Hermansky-Pudlak syndrome; Heterotaxy (X-linked visceral); Heterotopia(periventricular); Hippel-Lindau syndrom; Hirschsprung disease;Histidine-rich glycoprotein Thrombophilia due to HRG deficiency; HMG-CoAlyase deficiency; Holoprosencephaly-2; Holoprosencephaly-3;Holoprosencephaly-4; Holoprosencephaly-5; Holt-Oram syndrome;Homocystinuria; Hoyeraal-Hreidarsson; HPFH (deletion type or nondeletiontype); HPRT-related gout; Huntington disease; Hydrocephalus due toaqueductal stenosis; Hydrops fetalis; Hyperbetalipoproteinemia;Hypercholesterolemia, familial; Hyperferritinemia-cataract syndrome;Hyperglycerolemia; Hyperglycinemia; Hyperimmunoglobulinemia D andperiodic fever syndrome; Hyperinsulinism; Hyperinsulinism-hyperammonemiasyndrome; Hyperkalemic periodic paralysis; Hyperlipoproteinemia;Hyperlysinemia; Hypermethioninemia (persistent, autosomal, dominant, dueto methionine, adenosyltransferase I/III deficiency);Hyperornithinemia-hyperammonemiahomocitrullinemia syndrome;Hyperoxaluria; Hyperparathyroidism; Hyperphenylalaninemia due topterin-4acarbinolamine dehydratase deficiency; Hyperproinsulinemia;Hyperprolinemia; Hypertension; Hyperthroidism (congenital);Hypertriglyceridemia; Hypoalphalipoproteinemia; Hypobetalipoproteinemia;Hypocalcemia; Hypochondroplasia; Hypochromic microcytic anemia;Hypodontia; Hypofibrinogenemia; Hypoglobulinemia and absent B cells;Hypogonadism (hypergonadotropic); Hypogonadotropic (hypogonadism);Hypokalemic periodic paralysis; Hypomagnesemia; Hypomyelination(congenital); Hypoparathyroidism; Hypophosphatasia (adult; childhood;infantile; hereditary); Hypoprothrombinemia; Hypothyroidism (congenital;hereditary congenital; nongoitrous); Ichthyosiform erythroderma;Ichthyosis; Ichthyosis bullosa of Siemens; IgG2 deficiency; Immotilecilia syndrome-1; Immunodeficiency (T-cell receptor/CD3 complex);Immunodeficiency (X-linked, with hyper-IgM); Immunodeficiency due todefect in CD3-gamma; Immunodeficiency-centromeric instabilityfacialanomalies syndrome; Incontinentia pigmenti; Insensitivity to pain(congenital, with anhidrosis); Insomnia (fatal familial); Interleukin-2receptor deficiency (alpha chain); Intervertebral disc disease;Iridogoniodysgenesis; Isolated growth hormone deficiency (IIIig typewith absent GH and Kowarski type with bioinactive GH);Isovalericacidemia; Jackson-Weiss syndrome; Jensen syndrome; Jervell andLange-Nielsen syndrome; Joubert syndrom; Juberg-Marsidi syndrome;Kallmann syndrome; Kanzaki disease; Keratitis; Keratoderma(palmoplantar); Keratosis palmoplantaris striata I; Keratosispalmoplantaris striata II; Ketoacidosis due to SCOT deficiency; Keutelsyndrome; Klippel-Trenaumay syndrom; Kniest dysplasia; Kostmannneutropenia; Krabbe disease; Kurzripp-Polydaktylie syndrom;Lacticacidemia due to PDX1 deficiency; Langer mesomelic dysplasia; Larondwarfism; Laurence-Moon-Biedl-Bardet syndrom; LCHAD deficiency; Lebercongenital amaurosis; Left-right axis malformation; Leigh syndrome;Leiomyomatosis (diffuse, with Alport syndrome); Leprechaunism;Leri-Weill dyschondrosteosis; Lesch-Nyhan syndrome; Leukemia (acutemyeloid; acute promyelocytic; acute T-cell lymphoblastic; chronicmyeloid; juvenile myelomonocytic; Leukemia-1 (T-cell acute lymphocytic);Leukocyte adhesion deficiency; Leydig cell adenoma; Lhermitte-Duclossyndrome; Liddle syndrome; Li-Fraumeni syndrome; Lipoamide dehydrogenasedeficiency; Lipodystrophy; Lipoid adrenal hyperplasia; Lipoproteinlipase deficiency; Lissencephaly (X-linked); Lissencephaly-1; liverGlycogen storage disease (type 0); Long QT syndrome-1; Long QTsyndrome-2; Long QT syndrome-3; Long QT syndrome-5; Long QT syndrome-6;Lowe syndrome; Lung cancer; Lung cancer (nonsmall cell); Lung cancer(small cell); Lymphedema; Lymphoma (B-cell non-Hodgkin); Lymphoma(diffuse large cell); Lymphoma (follicular); Lymphoma (MALT); Lymphoma(mantel cell); Lymphoproliferative syndrome (X-linked); Lysinuricprotein intolerance; Machado-Joseph disease; Macrocytic anemiarefractory (of 5q syndrome); Macular dystrophy; Malignant mesothelioma;Malonyl-CoA decarboxylase deficiency; Mannosidosis, (alpha- or beta-);Maple syrup urine disease (type Ia; type Ib; type II); Marfan syndrome;Maroteaux-Lamy syndrome; Marshall syndrome; MASA syndrome; Mast cellleukemia; Mastocytosis with associated hematologic disorder; McArdledisease; McCune-Albright polyostotic fibrous dysplasia; McKusick-Kaufmansyndrome; McLeod phenotype; Medullary thyroid carcinoma;Medulloblastoma; Meesmann corneal dystrophy; Megaloblastic anemia-1;Melanoma; Membroproliferative glomerulonephritis; Meniere disease;Meningioma (NF2-related; SIS-related); Menkes disease; Mentalretardation (X-linked); Mephenytoin poor metabolizer; Mesothelioma;Metachromatic leukodystrophy; Metaphyseal chondrodysplasia (Murk Jansentype; Schmid type); Methemoglobinemia; Methionine adenosyltransferasedeficiency (autosomal recessive); Methylcobalamin deficiency (cbl Gtype); Methylmalonicaciduria (mutase deficiency type);Mevalonicaciduria; MHC class II deficiency; Microphthalmia (cataracts,and iris abnormalities); Miyoshi myopathy; MODY; Mohr-Tranebjaergsyndrome; Molybdenum cofactor deficiency (type A or type B);Monilethrix; Morbus Fabry; Morbus Gaucher; Mucopolysaccharidosis;Mucoviscidosis; Muencke syndrome; Muir-Torre syndrome; Mulibrey nanism;Multiple carboxylase deficiency (biotinresponsive); Multiple endocrineneoplasia; Muscle glycogenosis; Muscular dystrophy (congenitalmerosindeficient); Muscular dystrophy (Fukuyama congenital); Musculardystrophy (limb-girdle); Muscular dystrophy) Duchenne-like); Musculardystrophy with epidermolysis bullosa simplex; Myasthenic syndrome(slow-channel congenital); Mycobacterial infection (atypical, familialdisseminated); Myelodysplastic syndrome; Myelogenous leukemia; Myeloidmalignancy; Myeloperoxidase deficiency; Myoadenylate deaminasedeficiency; Myoglobinuria/hemolysis due to PGK deficiency;Myoneurogastrointestinal encephalomyopathy syndrome; Myopathy (actin;congenital; desmin-related; cardioskeletal; distal; nemaline); Myopathydue to CPT II deficiency; Myopathy due to phosphoglycerate mutasedeficiency; Myotonia congenita; Myotonia levior; Myotonic dystrophy;Myxoid liposarcoma; NAGA deficiency; Nailpatella syndrome; Nemalinemyopathy 1 (autosomal dominant); Nemaline myopathy 2 (autosomalrecessive); Neonatal hyperparathyroidism; Nephrolithiasis;Nephronophthisis (juvenile); Nephropathy (chronic hypocomplementemic);Nephrosis-1; Nephrotic syndrome; Netherton syndrome; Neuroblastoma;Neurofibromatosis (type 1 or type 2); Neurolemmomatosis; neuronal-5Ceroid-lipofuscinosis; Neuropathy; Neutropenia (alloimmune neonatal);Niemann-Pick disease (type A; type B; type C1; type D); Night blindness(congenital stationary); Nijmegen breakage syndrome; Noncompaction ofleft ventricular myocardium; Nonepidermolytic palmoplantar keratoderma;Norrie disease; Norum disease; Nucleoside phosphorylase deficiency;Obesity; Occipital hornsyndrome; Ocular albinism (Nettleship-Fallstype); Oculopharyngeal muscular dystorphy; Oguchi disease; Oligodontia;Omenn syndrome; Opitz G syndrome; Optic nerve coloboma with renaldisease; Ornithine transcarbamylase deficiency; Oroticaciduria;Orthostatic intolerance; OSMED syndrome; Ossification of posteriorlongitudinal ligament of spine; Osteoarthrosis; Osteogenesis imperfecta;Osteolysis; Osteopetrosis (recessive or idiopathic); Osteosarcoma;Ovarian carcinoma; Ovarian dysgenesis; Pachyonychia congenita(Jackson-Lawler type or Jadassohn-Lewandowsky type); Paget disease ofbone; Pallister-Hall syndrome; Pancreatic agenesis; Pancreatic cancer;Pancreatitis; Papillon-Lefevre syndrome; Paragangliomas; Paramyotoniacongenita; Parietal foramina; Parkinson disease (familial or juvenile);Paroxysmal nocturnal hemoglobinuria; Pelizaeus-Merzbacher disease;Pendred syndrome; Perineal hypospadias; Periodic fever; Peroxisomalbiogenesis disorder; Persistent hyperinsulinemic hypoglycemia ofinfancy; Persistent Mullerian duct syndrome (type II); Peters anomaly;Peutz-Jeghers syndrome; Pfeiffer syndrome; Phenylketonuria;Phosphoribosyl pyrophosphate synthetaserelated gout; Phosphorylasekinase deficiency of liver and muscle; Piebaldism; Pilomatricoma;Pinealoma with bilateral retinoblastoma; Pituitary ACTH secretingadenoma; Pituitary hormone deficiency; Pituitary tumor; Placentalsteroid sulfatase deficiency; Plasmin inhibitor deficiency; Plasminogendeficiency (types I and II); Plasminogen Tochigi disease; Plateletdisorder; Platelet glycoprotein IV deficiency; Platelet-activatingfactor acetylhydrolase deficiency; Polycystic kidney disease; Polycysticlipomembranous osteodysplasia with sclerosing leukenencephalophathy;Polydactyly, postaxial; Polyposis; Popliteal pterygium syndrome;Porphyria (acute hepatic or acute intermittent or congenitalerythropoietic); Porphyria cutanea tarda; Porphyriahepatoerythropoietic; Porphyria variegata; Prader-Willi syndrome;Precocious puberty; Premature ovarian failure; Progeria Typ I; ProgeriaTyp II; Progressive external ophthalmoplegia; Progressive intrahepaticcholestasis-2; Prolactinoma (hyperparathyroidism, carcinoid syndrome);Prolidase deficiency; Propionicacidemia; Prostate cancer; Protein Sdeficiency; Proteinuria; Protoporphyria (erythropoietic);Pseudoachondroplasia; Pseudohermaphroditism; Pseudohypoaldosteronism;Pseudohypoparathyroidism; Pseudovaginal perineoscrotal hypospadias;Pseudovitamin D deficiency rickets; Pseudoxanthoma elasticum (autosomaldominant; autosomal recessive); Pulmonary alveolar proteinosis;Pulmonary hypertension; Purpura fulminans; Pycnodysostosis;Pyropoikilocytosis; Pyruvate carboxylase deficiency; Pyruvatedehydrogenase deficiency; Rabson-Mendenhall syndrome; Refsum disease;Renal cell carcinoma; Renal tubular acidosis; Renal tubular acidosiswith deafness; Renal tubular acidosis-osteopetrosis syndrome;Reticulosis (familial histiocytic); Retinal degeneration; Retinaldystrophy; Retinitis pigmentosa; Retinitis punctata albescens;Retinoblastoma; Retinol binding protein deficiency; Retinoschisis; Rettsyndrome; Rh(mod) syndrome; Rhabdoid predisposition syndrome; Rhabdoidtumors; Rhabdomyosarcoma; Rhabdomyosarcoma (alveolar); Rhizomelicchondrodysplasia punctata; Ribbing-Syndrom; Rickets (vitaminD-resistant); Rieger anomaly; Robinow syndrome; Rothmund-Thomsonsyndrome; Rubenstein-Taybi syndrome; Saccharopinuria; Saethre-Chotzensyndrome; Salla disease; Sandhoff disease (infantile, juvenile, andadult forms); Sanfilippo syndrome (type A or type B); Schindler disease;Schizencephaly; Schizophrenia (chronic); Schwannoma (sporadic); SCID(autosomal recessive, T-negative/Bpositive type); Secretory pathwayw/TMD; SED congenita; Segawa syndrome; Selective T-cell defect; SEMD(Pakistani type); SEMD (Strudwick type); Septooptic dysplasia; Severecombined immunodeficiency (B cellnegative); Severe combinedimmunodeficiency (T-cell negative, B-celVnatural killer cell-positivetype); Severe combined immunodeficiency (Xlinked); Severe combinedimmunodeficiency due to ADA deficiency; Sex reversal (XY, with adrenalfailure); Sezary syndrome; Shah-Waardenburg syndrome; Short stature;Shprintzen-Goldberg syndrome; Sialic acid storage disorder; Sialidosis(type I or type II); Sialuria; Sickle cell anemia; Simpson-Golabi-Behmelsyndrome; Situs ambiguus; Sjogren-Larsson syndrome; Smith-Fineman-Myerssyndrome; Smith-Lemli-Opitz syndrome (type I or type II);Somatotrophinoma; Sorsby fundus dystrophy; Spastic paraplegia;Spherocytosis; Spherocytosis-1; Spherocytosis-2; Spinal and bulbarmuscular atrophy of Kennedy; Spinal muscular atrophy; Spinocerebellarataxia; Spondylocostal dysostosis; Spondyloepiphyseal dysplasia tarda;Spondylometaphyseal dysplasia (Japanese type); Stargardt disease-1;Steatocystoma multiplex; Stickler syndrome; Sturge-Weber syndrom;Subcortical laminal heteropia; Subcortical laminar heterotopia; Succinicsemialdehyde dehydrogenase deficiency; Sucrose intolerance;Sutherland-Haan syndrome; Sweat chloride elevation without CF;Symphalangism; Synostoses syndrome; Synpolydactyly; Tangier disease;Tay-Sachs disease; T-cell acute lymphoblastic leukemia; T-cellimmunodeficiency; T-cell prolymphocytic leukemia; Thalassemia (alpha- ordelta-); Thalassemia due to Hb Lepore; Thanatophoric dysplasia (types Ior II); Thiamine-responsive megaloblastic anemia syndrome;Thrombocythemia; Thrombophilia (dysplasminogenemic); Thrombophilia dueto heparin cofactor II deficiency; Thrombophilia due to protein Cdeficiency; Thrombophilia due to thrombomodulin defect; Thyroid adenoma;Thyroid hormone resistance; Thyroid iodine peroxidase deficiency; Tietzsyndrome; Tolbutamide poor metabolizer; Townes-Brocks syndrome;Transcobalamin II deficiency; Treacher Collins mandibulofacialdysostosis; Trichodontoosseous syndrome; Trichorhinophalangeal syndrome;Trichothiodystrophy; Trifunctional protein deficiency (type I or typeII); Trypsinogen deficiency; Tuberous sclerosis-1; Tuberous sclerosis-2;Turcot syndrome; Tyrosine phosphatase; Tyrosinemia; Ulnar-mammarysyndrome; Urolithiasis (2,8-dihydroxyadenine); Usher syndrome (type 1Bor type 2A); Venous malformations; Ventricular tachycardia;Virilization; Vitamin K-dependent coagulation defect; VLCAD deficiency;Vohwinkel syndrome; von Hippel-Lindau syndrome; von Willebrand disease;Waardenburg syndrome; Waardenburg syndrome/ocular albinism;Waardenburg-Shah neurologic variant; Waardenburg-Shah syndrome; Wagnersyndrome; Warfarin sensitivity; Watson syndrome;Weissenbacher-Zweymuller syndrome; Wemer syndrome; Weyers acrodentaldysostosis; White sponge nevus; Williams-Beuren syndrome; Wilms tumor(type1); Wilson disease; Wiskott-Aldrich syndrome; Wolcott-Rallisonsyndrome; Wolfram syndrome; Wolman disease; Xanthinuria (type I);Xeroderma pigmentosum; X-SCID; Yemenite deaf-blind hypopigmentationsyndrome; ypocalciuric hypercalcemia (type I); Zellweger syndrome;Zlotogora-Ogur syndrome.

Preferred diseases to be treated which have a genetic inheritedbackground and which are typically caused by a single gene defect andare inherited according to Mendel's laws are preferably selected fromthe group consisting of autosomal-recessive inherited diseases, such as,for example, adenosine deaminase deficiency, familialhypercholesterolaemia, Canavan's syndrome, Gaucher's disease, Fanconianaemia, neuronal ceroid lipofuscinoses, mucoviscidosis (cysticfibrosis), sickle cell anaemia, phenylketonuria, alcaptonuria, albinism,hypothyreosis, galactosaemia, alpha-1-anti-trypsin deficiency, Xerodermapigmentosum, Ribbing's syndrome, mucopolysaccharidoses, cleft lip, jaw,palate, Laurence Moon Biedl Bardet sydrome, short rib polydactyliasyndrome, cretinism, Joubert's syndrome, type II progeria,brachydactylia, adrenogenital syndrome, and X-chromosome inheriteddiseases, such as, for example, colour blindness, e.g. red/greenblindness, fragile X syndrome, muscular dystrophy (Duchenne andBecker-Kiener type), haemophilia A and B, G6PD deficiency, Fabry'sdisease, mucopolysaccharidosis, Norrie's syndrome, Retinitis pigmentosa,septic granulomatosis, X-SCID, ornithine transcarbamylase deficiency,Lesch-Nyhan syndrome, or from autosomal-dominant inherited diseases,such as, for example, hereditary angiooedema, Marfan syndrome,neurofibromatosis, type I progeria, Osteogenesis imperfecta,Klippel-Trenaurnay syndrome, Sturge-Weber syndrome, Hippel-Lindausyndrome and tuberosis sclerosis.

The present invention also allows treatment of diseases, which have notbeen inherited, or which may not be summarized under the abovecategories. Such diseases may include e.g. the treatment of patients,which are in need of a specific protein factor, e.g. a specifictherapeutically active protein as mentioned above. This may e.g. includedialysis patients, e.g. patients which undergo a (regular) a kidney orrenal dialysis, and which may be in need of specific therapeuticallyactive proteins as defined above, e.g. erythropoietin (EPO), etc.

According to another embodiment, the present invention comprises the useof the at least one complexed RNA according to the present invention fortransfecting a cell or an organism. Transfection of the cell or theorganism may preferably be carried out using the above (in vitro or invivo) transfection method for transfecting cells or a tissue with thecomplexed RNA of the present invention.

According to one further embodiment, the present invention comprises theuse of at least one complexed RNA according to the present invention(for the preparation of an agent) for the treatment of any of the abovementioned diseases, disorders, conditions or pathological states. Anagent in this context may be e.g. a pharmaceutical composition asdefined above or an injection buffer as defined herein, additionallycontaining the inventive complexed RNA, a vaccine, etc. If more than onecomplexed RNA molecule type is used, the complexed RNAs may be differentby their RNA (molecules) thereby forming a mixture of at least twodistinct complexed RNA (molecule) types. If more than one complexed RNAis used (for the preparation of an agent) for the treatment of any ofthe above mentioned diseases the same or (at least two) different RNA(molecule) types may be contained in these complexed RNA mixtures. Inthis context, any of the above mentioned RNA (molecules) may be used forthe inventive complexed RNA, e.g. a short RNA oligonucleotide, a codingRNA, an immunostimulatory RNA, a siRNA, an antisense RNA, orriboswitches, ribozymes or aptamers, etc. More preferably a coding RNA(molecule), even more preferably a linear coding RNA (molecule), andmost preferably an mRNA may be used. Preferably, such a coding RNA(molecule), more preferably a linear coding RNA (molecule), and morepreferably an mRNA is used for the complexed RNA, the RNA (molecule)typically encodes a protein or peptide suitable for the therapy of thespecific disease, e.g. an antibody, which is capable of binding to aspecific cancer antigen, or a tumor antigen, when treating a (specific)cancer, etc. The combinations of suitable RNA (molecules) are known to askilled person from the art and from the disclosure of the presentinvention.

According to another embodiment of the present invention, it may bepreferred to (additionally) elicit, e.g. induce or enhance, an immuneresponse during therapy. In this context, an immune response may occurin various ways. A substantial factor for a suitable immune response isthe stimulation of different T-cell sub-populations. T-lymphocytes aretypically divided into two sub-populations, the T-helper 1 (Th1) cellsand the T-helper 2 (Th2) cells, with which the immune system is capableof destroying intracellular (Th1) and extracellular (Th2) pathogens(e.g. antigens). The two Th cell populations differ in the pattern ofthe effector proteins (cytokines) produced by them. Thus, Th1 cellsassist the cellular immune response by activation of macrophages andcytotoxic T-cells. Th2 cells, on the other hand, promote the humoralimmune response by stimulation of the B-cells for conversion into plasmacells and by formation of antibodies (e.g. against antigens). TheTh1/Th2 ratio is therefore of major importance for the immune response.For various diseases to be treated by the present invention, the Th1/Th2ratio of the immune response is preferably shifted in the directiontowards the cellular response (Th1 response) and a cellular immuneresponse is thereby induced. Accordingly, the present invention may alsobe used to revert this immune response shift. Therefore, the presentinvention encompasses also the use of at least one complexed RNAaccording to the present invention (for the preparation of an agent) forthe treatment of any of the above mentioned diseases, wherein the agent(and/or the complexed RNA) may be capable to elicit, e.g. induce orenhance, an immune response in a tissue or an organism as defined above.Again, an agent in this context may be e.g. a pharmaceutical compositionas defined above, or an injection buffer as defined herein, whichcontains the inventive complexed RNA, etc. If more than one complexedRNA type is used in this context (for the preparation of an agent) forthe treatment of any of the above mentioned diseases, the complexed RNAtypes may be different with respect to their RNA (molecules) and mayform a mixture of distinct RNA types.

However, for the present embodiment, it is preferred that at least oneof these complexed RNAs induces or enhances the immune response duringtherapy, while other complexed RNA(s) need not to induce or enhance theimmune response or may be used to prevent an immune response. In thiscontext, any of the above mentioned RNA (molecules) may be used for theinventive complexed RNA, e.g. a short RNA oligonucleotide, a coding RNA,an immunostimulatory RNA, a siRNA, an antisense RNA, or riboswitches,ribozymes or aptamers, etc. More preferably, a coding RNA (molecule),even more preferably a linear coding RNA (molecule), and most preferablyan mRNA may be used for the complexed RNA. If the RNA (molecule) is acoding RNA (molecule), more preferably a linear coding RNA (molecule),and more preferably an mRNA, it typically encodes a protein or peptidesuitable for the therapy of the specific disease, e.g. an antibody,which is capable of binding to a specific cancer antigen, when treatinga (specific) cancer, etc. If more than one complexed RNA is contained inthe agent, different combinations of proteins or peptides may beselected. Such combinations of suitable RNA (molecules) (and, if acoding RNA is used, of encoded proteins or peptides) are known to askilled person from the art or may be combined from RNAs encodingtherapeutically effective proteins, etc., as defined in the disclosureof the present invention. Induction or enhancement of the immuneresponse concurrent to the treatment of a specific disease using onepharmaceutical composition or agent as defined above may be particularlyadvantageous in cases where an induced or enhanced immune responsesupports the treatment of a specific disease as mentioned above.

Alternatively, treatment of the disease and induction or enhancement ofthe immune response may be carried out by using different pharmaceuticalcompositions or agents as defined above in a time staggered manner. E.g.one may induce or enhance the immune response by administering apharmaceutical composition or an agent as defined herein, containing aninventive complexed (immunostimulatory) RNA, prior to (or concurrent to)administering another pharmaceutical composition or an agent as definedherein which may contain an inventive complexed RNA, e.g. a short RNAoligonucleotide, a coding RNA, an immunostimulatory RNA, a siRNA, anantisense RNA, or riboswitches, ribozymes or aptamers, etc., which issuitable for the therapy of the specific disease.

According to one embodiment, the present invention furthermore comprisesthe use of at least one complexed RNA according to the present invention(for the preparation of an agent) for modulating, preferably to induceor enhance, an immune response in a tissue or an organism as definedabove, more preferably to support a disease or state as mentionedherein. Hereby, the inventive complexed RNA may be used to activate theimmune system unspecifically, e.g. to trigger the production of certaincytokines. The complexed RNA may therefore be used to support thespecific immune response, which is elicited by e.g. an antigen derivedfrom pathogens or tumors. An agent in this context may be e.g. apharmaceutical composition as defined above or an injection buffer asdefined herein, containing the inventive complexed RNA, a vaccine, etc.The immune response may be modulated either by the at least onecomplexed RNA due to the one or more oligopeptides having a length of 8to 15 amino acids and showing the empirical formula(Arg)_(l);(Lys)_(m);(His)_(n);(Orn)_(o);(Xaa)_(x), and/or by theimmunostimulatory properties of the protein encoded by the RNA of thecomplexed RNA.

The present invention may therefore, whenever appropriate, well serve toachieve various objects. A complexed RNA as such or as a component of aninventive composition may by itself improve the transfection propertiesof the RNA as component of the inventive complex. This underlyingproperty of the inventive complexed RNA is beneficial to a wide varietyof applications. Whenever it is intended to introduce an RNA into acell, improved transfection efficacy is ensured by the presentinvention. This property as such may allow the present invention to beused for the treatment of a huge variety of diseases, e.g. the treatmentof monogenetic or genetic diseases as defined above.

In addition, the present invention may be used whenever treatment ofimmune disorders, e.g. allergies or autoimmune diseases, is envisaged.Moreover, the present invention may activate the patients's immunesystem by enhancing its unspecific or specific immune response.Accordingly, it may elicit an unspecific immune response, wheneverappropriate, to cure a disease. And, whenever required, it may elicit aspecific immune response as such (e.g. by encoding an antigen by the RNAas component of the inventive complex) or by a combination of theinventive complexed RNA with an antigen, e.g. in the same composition.Whenever required, the inventive complexed RNA may be preferably anantigen or an antibody, or any other protein or peptide as definedabove, capable of modulating the immune response (preferably of inducingor enhancing same or, in case of allergies or autoimmune diseases bydesensitizing the patient's immune system towards a specific allergen orautoantigen). In order to modulate, e.g. induce or enhance, an immuneresponse in a tissue or an organism the complexed RNA may beadministered to this tissue or organism as defined above either as suchor as an agent as defined above. The administration modes, which may beused, may be the same as described above for pharmaceuticalcompositions. Administration of the agent may occur prior, concurrentand/or subsequent to a therapy of diseases or states as mentionedherein, e.g. by administration of the agent prior, concurrent and/orsubsequent to a therapy or an administration of a therapeutic suitablefor these diseases or states.

According to an alternative embodiment, the present invention alsoencompasses the use of the peptide (Arg)₇ in a complex with an RNA(molecule) as defined herein or the peptide (Arg)₇ alone (for thepreparation of an agent) for modulating, preferably to elicit, e.g. toinduce or enhance, an immune response, preferably an unspecific immuneresponse by e.g. triggering the production of cytokines, in a tissue oran organism as defined above, and preferably to support a disease orstate as mentioned herein. While determining the ranges of the peptideof the inventive formula (I), the present inventors have surprisinglyfound that (Arg)₇ is capable to significantly induce or enhance animmune response in hPBMCs, even if no transfection of nucleic acids,particularly RNA into hPBMCs, was observed. An RNA (molecule) may be anyRNA (molecule) as defined herein, preferably, without being limitedthereto, a short RNA oligonucleotide, a coding RNA, an immunostimulatoryRNA, a siRNA, an antisense RNA, or riboswitches, ribozymes or aptamers.Again, an agent in this context may be e.g. a pharmaceutical compositionas defined above, or an injection buffer as defined herein, whichadditionally contains the inventive complexed RNA, etc., wherein in theinventive complexed RNA in the agent as defined herein has been replacedby the peptide (Arg)₇ in a complex with an RNA (molecule) as definedherein or the peptide (Arg)₇ alone.

According to another alternative embodiment, the present invention alsoencompasses the use of the peptide (Arg)₇ in a complex with an RNA(molecule) as defined herein or the peptide (Arg)₇ alone (for thepreparation of an agent) for the treatment of any of the above mentioneddiseases or states.

According to a final embodiment, the present invention also provideskits containing a complexed RNA according to the invention and/or apharmaceutical composition according to the invention as well as,optionally, technical instructions with information on theadministration and dosage of the complexed RNA according to theinvention and/or the pharmaceutical composition according to theinvention. The kit may separately further comprise one or more of thefollowing group of components: at least one antigen or at least oneantibody or a composition containing an antigen or an antibody, anadditional adjuvant or a composition containing at least one adjuvantand/or at least one cytokine or a composition containing at least onecytokine. The antigen, antibody and/or the cytokine may be provided assuch (proteins) or may be provided as DNA or RNA coding for the antigen,antibody or cytokine.

The present invention also provides kits containing the peptide (Arg)₇in a complex with an RNA (molecule) as defined herein or the peptide(Arg)₇ alone as well as, optionally, technical instructions withinformation on the administration and dosage of the peptide (Arg)₇. Suchkits may applied e.g. for any of the above mentioned applications oruses, preferably for the use of at least one complexed RNA according tothe present invention (for the preparation of an agent) for thetreatment of any of the above mentioned diseases. The kits may also beapplied for the use of at least one complexed RNA according to thepresent invention (for the preparation of an agent) for the treatment ofany of the above mentioned diseases, wherein the agent (and/or thecomplexed RNA) may be capable to induce or enhance an immune response ina tissue or an organism as defined above. Such kits may further beapplied for the use of at least one complexed RNA according to thepresent invention (for the preparation of an agent) for modulating,preferably to elicit, e.g. to induce or enhance, an immune response in atissue or an organism as defined above, and preferably to support adisease or state as mentioned herein.

FIGURES

The following Figures are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

FIG. 1: depicts the sequence of a stabilized luciferase mRNA sequence,wherein the native luciferase encoding mRNA is modified with apoly-A/poly-C-tag (A70-C30). This first construct (constructCAP-Ppluc(wt)-muag-A70-C30, SEQ ID NO: 35) contained following sequenceelements:

-   -   stabilizing sequences from the alpha-Globin gene,    -   70× Adenosin at the 3′-terminal end (poly-A-tail),    -   30× Cytosin at the 3′-terminal end (poly-C-tail);    -   represented by following symbols:    -   =coding sequence    -   =3′-UTR of the alpha globin gene    -   =poly-A-tail    -   =poly-C-tail

FIG. 2: shows the sequence of a stabilized luciferase mRNA sequence,wherein the construct according to SEQ ID NO: 35 (see FIG. 1) is furthermodified with a GC-optimized sequence for a better codon usage. Thefinal construct (construct CAP-Ppluc(GC)-muag-A70-C30, SEQ ID NO: 36)contained following sequence elements:

-   -   GC-optimized sequence for a better codon usage    -   stabilizing sequences from the alpha-Globin gene    -   70× Adenosin at the 3′-terminal end (poly-A-tail),    -   30× Cytosin at the 3′-terminal end (poly-C-tail);    -   represented by following symbols:    -   =coding sequence    -   =modified 3′-UTR of the alpha globin gene    -   =poly-A-tail    -   =poly-C-tail

FIG. 3: shows the coding sequence of the sequence according to SEQ IDNO: 35 (SEQ ID NO: 37) (see FIG. 1).

FIG. 4: shows the GC-optimized coding sequence of the sequence accordingto SEQ ID NO: 36 (SEQ ID NO: 38) (see FIG. 2). The GC-optimized codonsare underlined.

FIG. 5: shows the immunostimulatory effect of RNA complexed withnona-arginine ((Arg)₉) in hPBMC cells by measuring IL-6 production. Ascan be seen, hPBMC cells show a significant IL-6 production, i.e. asignificant immunostimulatory effect of RNA complexed with nona-arginine((Arg)₉).

FIG. 6: shows the immunostimulatory effect of RNA complexed withnona-arginine ((Arg)₉) in hPBMC cells by measuring TNF-alpha production.As can be seen, hPBMC cells show a significant TNF-alpha production,i.e. a significant immunostimulatory effect of RNA complexed withnona-arginine ((Arg)₉).

FIG. 7: shows in an comparative example the comparison ofimmunostimulatory effects of RNA complexed with either nona-arginine((Arg)₉) or poly-L-arginine, respectively, in hPBMCs. Advantageously, asignificant immunostimulatory effect can be observed for mass ratioslower than 1:5 (RNA:nona-arginine) (1:10; 1:8; 1:5; 1:2; 1:1; 2:1).However, when using mass ratios of RNA:nona-arginine (5:1) nosignificant TNFalpha production can be observed. The same applies tostimulation experiments, using nona-arginine ((Arg)₉) or mRNA alone.Additionally, it was observed, that complexation of mRNA withpoly-L-arginine leads to significantly lower induction of TNF-alphaproduction in comparison to nona-arginine ((Arg)₉). Apparently, higherconcentrations of poly-L-arginine appear to be toxic for cellstransfected therewith, particularly when using a mass ratio of 1:2RNA:poly-L-arginine:RNA or higher, since the cells were lysed.

FIG. 8: shows luciferase expression upon transfection of complexes ofRNA with nona-arginine ((Arg)₉) in HeLa cells. As may be derived fromFIG. 8 a mass ratio of less than 2:1 (RNA:nona-arginine) appears to beadvantageous. In contrast, complexation with (high molecular mass)poly-L-arginine does not lead to a significant luciferase-activity.Thus, (high molecular mass) poly-L-arginine does not appear to besuitable for transfection of mRNA.

FIG. 9: depicts in a comparative example the luciferase expression upontransfection of complexes of RNA with hepta-arginine ((Arg)₇) in HeLacells. As may be derived from FIG. 9, transfection of complexes of RNAwith hepta-arginine ((Arg)₇) does not lead to a significantluciferase-activity. Thus, hepta-arginine ((Arg)₇) does also not appearto be suitable for transfection of mRNA.

FIG. 10: shows the immunostimulatory effect of RNA complexed withhepta-arginine ((Arg)₇) in hPBMC cells by measuring IL-6 production. Ascan be seen, hPBMC cells show a significant IL-6 production, i.e. asignificant immunostimulatory effect of RNA complexed withhepta-arginine ((Arg)₇).

FIG. 11: shows the immunostimulatory effect of RNA complexed withhepta-arginine ((Arg)₇) in hPBMC cells by measuring TNF-alphaproduction. As can be seen, hPBMC cells show a significant TNF-alphaproduction, i.e. a significant immunostimulatory effect of RNA complexedwith hepta-arginine ((Arg)₇).

FIG. 12: shows the effect of RNA complexed with R9 peptide on theexpression of luciferase in HeLa cells.

FIG. 13: shows the effect of RNA complexed with R9H3 peptide on theexpression of luciferase in HeLa cells.

FIG. 14: shows the effect of RNA complexed with H3R9H3 peptide on theexpression of luciferase in HeLa cells.

FIG. 15: shows the effect of RNA complexed with YYYR9SSY peptide on theexpression of luciferase in HeLa cells.

FIG. 16: shows the effect of RNA complexed with H3R9SSY peptide on theexpression of luciferase in HeLa cells.

FIG. 17: shows the effect of RNA complexed with (RKH)4 peptide on theexpression of luciferase in HeLa cells.

FIG. 18: shows the effect of RNA complexed with Y(RKH)2R peptide on theexpression of luciferase in HeLa cells.

FIG. 19: shows the effect of Histidin in terminal positions on thetransfection efficacy.

FIG. 20: shows the effect of neutral amino acids in terminal positionson the transfection efficacy.

FIG. 21: shows the immunostimulatory effect of RNA complexed with R9H3on secretion of TNFalpha in hPBMCs.

FIG. 22: shows the immunostimulatory effect of RNA complexed with R9H3on secretion of IL-6 in hPBMCs.

EXAMPLES

The following examples are intended to illustrate the invention further.They are not intended to limit the subject matter of the inventionthereto.

Example 1—Preparation of Luciferase mRNA Constructs

In the following experiments a stabilized luciferase mRNA sequence wasprepared and used for transfection experiments, wherein the nativeluciferase encoding mRNA was modified with a poly-A/poly-C-tag (A70-C30)and was GC-optimized for a better codon-usage and further stabilized.

A first construct (construct CAP-Ppluc(wt)-muag-A70-C30, SEQ ID NO: 35)contained following sequence elements:

-   -   stabilizing sequences from the alpha-Globin gene    -   70 (Adenosin at the 3′-terminal end    -   30 (Cytosin at the 3′-terminal end

The final construct (construct CAP-Ppluc(GC)-muag-A70-C30, SEQ ID NO:36), as used herein for the following experiments, contained followingsequence elements:

-   -   GC-optimized sequence for a better codon usage    -   stabilizing sequences from the alpha-Globin gene    -   70 (Adenosin at the 3′-terminal end    -   30 (Cytosin at the 3′-terminal end

These sequences are also shown in FIGS. 1 and 2 (SEQ ID NOs: 35 and 36).The respective coding sequences are shown in FIGS. 3 and 4 (SEQ ID NOs:35 and 36)

Example 2—In Vitro Transcription of Stabilized Luciferase mRNA

The stabilized luciferase mRNA according to SEQ ID NO: 35 or 36(Luc-RNActive) was transcribed in vitro using T7-Polymerase (T7-OptimRNA Kit, CureVac, Tübingen, Deutschland) following the manufacturesinstructions.

All mRNA-transkripts contained a 70 bases poly-A-tail and a5′-Cap-structure. The 5′-Cap-structure was obtained by adding an excessof N7-Methyl-Guanosin-5′-Triphosphat-5′-Guanosin.

Example 3—Forming a Complex of RNA with Nona-Arginine ((Arg)₉),Poly-L-Arginine or Further Peptides Based on (Arg)₉ Respectively

Ng RNA stabilized luciferase mRNA according to SEQ ID NO: 36(Luc-RNActive) were mixed in different mass ratios with nona-arginine(Arg₉) or poly-L-arginine (Sigma-Aldrich; P4663; 5000-15000 g/mol),thereby forming a complex. Following mass ratios were used as shownexemplarily for ((Arg)₉). Poly-L-arginine was used for comparativeexamples following the same instructions.

(Arg)₉ (Arg)₉ RNA (Arg)₉ H₂0 Concentration Ratio RNA (Arg)₉ μg μg μl μlμl (Arg)₉ [μM] (Arg)₉/RNA 1 Mock 70.0 0 2 (Arg)₉ alone 150 3 67.0 151.323 RNA alone 15 3.8 66.3 0.00 4 1 10 15 150.0 3.8 3.0 63.3 151.32 10:1  51 8 15 120.0 3.8 2.4 63.9 121.06 8:1 6 1 5 15 75.0 3.8 1.5 64.8 75.665:1 7 1 2 15 30.0 3.8 0.6 65.7 30.26 2:1 8 1 1 15 15.0 3.8 15.0 51.315.13 1:1 9 2 1 15 7.5 3.8 7.5 58.8 7.57 1:2 10 5 1 15 3.0 3.8 3.0 63.33.03 1:5 11 8 1 15 1.9 3.8 1.9 64.4 1.89 1:8 12 10 1 15 1.5 3.8 1.5 64.81.51  1:10

Additionally, further complexed RNAs based on (Arg)₉ were prepared aboveusing the following peptides for complexation:

R9: Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg R9H3:Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-His-His-His H3R9H3:His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- His-His-His YSSR9SSY:Tyr-Ser-Ser-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr H3R9SSY:His-His-His-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg-Arg- Ser-Ser-Tyr (RKH)4:Arg-Lys-His-Arg-Lys-His-Arg-Lys-His-Arg-Lys-His Y(RKH)2R:Tyr-Arg-Lys-His-Arg-Lys-His-Arg

For complexation, 4 μg stabilized luciferase mRNA according to SEQ IDNO: 36 (Luc-RNActive) were mixed in molar ratios with the respectivelypeptide (according to formula I), thereby forming a complex. Afterwardsthe resulting solution was adjusted with water to a final volume of 50μl und incubated for 30 minutes at room temperature. The used ratios areindicated in the tables given below. HeLa-cells (150×10³/well) were thenseeded 1 day prior to transfection on 24-well microtiter plates leadingto a 70% confluence when transfection was carried out.

R9: Formulation −> N/P R9 Molar ratio Mass ratio RNA R9 RNA μg Peptid μgN/P 1.00 10000.00 1.00 23.72 50.00 1.00 5000.00 1.00 11.86 25.00 1.002500.00 1.00 5.93 12.50 1.00 1000.00 1.00 2.37 5.00 1.00 500.00 1.001.19 2.50 1.00 100.00 1.00 0.24 0.50 1.00 10.00 1.00 0.02 0.05

R9H3: Formulation −> N/P R9H3 Molar ratio Mass ratio RNA R9H3 RNA μgPeptid μg N/P 1.00 10000.00 1.00 30.58 50.00 1.00 5000.00 1.00 15.2925.00 1.00 2500.00 1.00 7.65 12.50 1.00 1000.00 1.00 3.06 5.00 1.00500.00 1.00 1.53 2.50 1.00 100.00 1.00 0.31 0.50 1.00 10.00 1.00 0.030.05

H3R9H3: Formulation −> N/P H3R9H3 Molar ratio Mass ratio RNA H3R9H3 RNAμg Peptid μg N/P 1.00 10000.00 1.00 37.43 50.00 1.00 5000.00 1.00 18.7225.00 1.00 2500.00 1.00 9.36 12.50 1.00 1000.00 1.00 3.74 5.00 1.00500.00 1.00 1.87 2.50 1.00 100.00 1.00 0.37 0.50 1.00 10.00 1.00 0.040.05

YSSR9SSY: YSSR9SSY YSSR9SSY Molar ratio Mass ratio RNA YSSR9SSY RNA μgPeptid μg N/P 1.00 10000.00 1.00 34.95 50.00 1.00 5000.00 1.00 17.4825.00 1.00 2500.00 1.00 8.74 12.50 1.00 1000.00 1.00 3.50 5.00 1.00500.00 1.00 1.75 2.50 1.00 100.00 1.00 0.35 0.50 1.00 10.00 1.00 0.030.05

H3R9SSY: Formulation −> N/P H3R9SSY Molar ratio Mass ratio RNA H3R9SSYRNA μg Peptid μg N/P 1.00 10000.00 1.00 36.18 50.00 1.00 5000.00 1.0018.09 25.00 1.00 2500.00 1.00 9.05 12.50 1.00 1000.00 1.00 3.62 5.001.00 500.00 1.00 1.81 2.50 1.00 100.00 1.00 0.36 0.50 1.00 10.00 1.000.04 0.05

(RKH)4: Formulation −> N/P (RKH)4 Molar ratio Mass ratio RNA (RKH)4 RNAμg Peptid μg N/P 1.00 10000.00 1.00 28.38 44.44 1.00 5000.00 1.00 14.1922.22 1.00 2500.00 1.00 7.10 11.11 1.00 1000.00 1.00 2.84 4.44 1.00500.00 1.00 1.42 2.22 1.00 100.00 1.00 0.28 0.44 1.00 10.00 1.00 0.030.04

Y(RKH)2R: Formulation −> N/P Y(RKH)2R Molar ratio Mass ratio RNAY(RKH)2R RNA μg Peptid μg N/P 1.00 10000.00 1.00 19.67 27.78 1.005000.00 1.00 9.83 13.89 1.00 2500.00 1.00 4.92 6.94 1.00 1000.00 1.001.97 2.78 1.00 500.00 1.00 0.98 1.39 1.00 100.00 1.00 0.20 0.28 1.0010.00 1.00 0.02 0.03

Example 4—Nona-Arginine((Arg)₉)-Mediated Transfection and Expression ofStabilized Luciferase mRNA According to SEQ ID NO: 35 or 36(Luc-RNActive) in HeLa-Cells

Hela-cells (150×10³/well) were seeded 1 day prior to transfection on24-well microtiter plates leading to a 70% confluence when transfectionwas carried out. For transfection (40 μl) 50 μl of theRNA/(peptide)-solution as disclosed in Example 3 were mixed with 250 μlserum free medium and added to the cells (final RNA concentration: 13μg/ml). Prior to addition of the transfection solution the HeLa-cellswere washed gently and carefully 2 times with 1 ml Optimen (Invitrogen)per well. Then, the transfection solution (300 μl per well) was added tothe cells and the cells were incubated for 4 h at 37° C. Subsequently300 μl RPMI-medium (Camprex) containing 10% FCS was added per well andthe cells were incubated for additional 20 h at 37° C. The transfectionsolution was sucked off 24 h after transfection and the cells were lysedin 300 μl lysis buffer (25 mM Tris-PO₄, 2 mM EDTA, 10% glycerol, 1%Triton-X 100, 2 mM DTT). The supernatants were then mixed with luciferinbuffer (25 mM Glycylglycin, 15 mM MgSO₄, 5 mM ATP, 62.5 μM luciferin)and luminiscence was detected using a luminometer (Lumat LB 9507(Berthold Technologies, Bad Wildbad, Germany)). The results of theseexperiments are shown in FIGS. 8 and 12 to 18.

Example 5—Immune Stimulation Upon Transfection of Complexes of RNA withNona-Arginine ((Arg)₉) or Poly-L-Arginine (Comparative Example) a)Transfection Experiments

-   -   HPBMC cells from peripheral blood of healthy donors were        isolated using a Ficoll gradient and washed subsequently with        1×PBS (phophate-buffered saline). The cells were then seeded on        96-well microtiter plates (200×103/well). The hPBMC cells were        incubated for 24 h, as described under Example 4, supra, with 10        μl of the RNA/peptide complex (RNA final concentration: 6 μg/ml;        the same amounts of RNA were used) in X-VIVO 15 Medium        (BioWhittaker) (final RNA Concentration: 10 μg/ml). The        immunostimulatory effect upon the hPBMC cells was measured by        detecting the cytokine production (Interleukin-6 und Tumor        necrose factor alpha). Therefore, ELISA microtiter plates (Nunc        Maxisorb) were incubated over night (o/n) with binding buffer        (0.02% NaN3, 15 mM Na2CO3, 15 mM NaHCO3, pH 9.7), additionally        containing a specific cytokine antibody. Cells were then blocked        with 1×PBS, containing 1% BSA (bovine serum albumin). The cell        supernatant was added and incubated for 4 h at 37° C.        Subsequently, the microtiter plate was washed with 1×PBS, 0.05%        Tween-20 and then incubated with a Biotin-labelled secondary        antibody (BD Pharmingen, Heidelberg, Germany).        Streptavidin-coupled horseraddish peroxidase was added to the        plate. Then, the plate was again washed with 1×PBS, containing        0.05% Tween-20, and ABTS        (2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was        added as a substrate. The amount of cytokine was determined by        measuring the absorption at 405 nm (OD405) using a standard        curve with recombinant Cytokines (BD Pharmingen, Heidelberg,        Germany) with the Sunrise ELISA-Reader from Tecan (Crailsheim,        Germany).

b) Results

i) Immunostimulatory Effect of RNA Complexed with Nona-Arginine ((Arg))

-   -   i1) HPBMC cells were incubated with RNA complexed with        nona-arginine ((Arg)₉) for 24 h as disclosed above, wherein the        mass ratio of RNA:(Arg)₉ was 1:1. Then, IL-6 production was        measured in the cell supernatants using ELISA. As a result,        HPBMC cells showed a significant IL-6 production, i.e. a        significant immunostimulatory effect of RNA complexed with        nona-arginine ((Arg)₉) (see FIG. 5).    -   i2) HPBMC cells were incubated with RNA complexed with        nona-arginine ((Arg)₉) for 24 h as disclosed above, wherein the        mass ratio of RNA:(Arg)₉ was 1:1. Then, THF-alpha production was        measured in the cell supernatants using ELISA. As a result,        HPBMC cells showed a significant TNF-alpha production, i.e. a        significant immunostimulatory effect of RNA complexed with        nona-arginine ((Arg)₉) (see FIG. 6).

ii) Comparison of Immunostimulatory Effect of RNA Complexed with EitherNona-Arginine ((Arg)₉) or Poly-L-Arginine, Respectively (ComparativeExample)

-   -   hPBMCs were incubated in different mass ratios        (RNA:nona-arginine 1:10; 1:8; 1:5; 1:2; 1:1; 2:1, 5:1; 8:1 and        10:1) with a complex of RNA and nona-arginine ((Arg)₉) or        poly-L-arginine, etc., respectively, for 24 h. Subsequently        TNF-alpha production was measured using ELISA.    -   Advantageously, a significant immunostimulatory effect can be        observed for mass ratios lower than 5:1 (RNA:nona-arginine)        (1:10; 1:8; 1:5; 1:2; 1:1; 2:1) (see FIG. 7). When using mass        ratios of RNA:nona-arginine (5:1) no significant TNFalpha        production can be observed. The same applies to stimulation        experiments, using nona-arginine ((Arg)₉) or mRNA alone (see        FIG. 7, left).    -   Furthermore, complexation of mRNA with poly-L-arginine leads to        significantly lower induction of TNF-alpha production in        comparison to nona-arginine ((Arg)₉) (see FIG. 7, right).        Additionally, it was observed that higher concentrations of        poly-L-arginine appear to be toxic for cells transfected        therewith, particularly when using a mass ratio of 1:2        RNA:poly-L-arginine or lower, since the cells were lysed.

Example 6—Luciferase Expression Upon Transfection of Complexes of RNAwith Nona-Arginine ((Arg)₉) or Poly-L-Arginine. Respectively, in HeLaCells (Comparative Example)

-   a) Luciferase expression upon transfection of complexes of RNA with    nona-arginine ((Arg)₉) in HeLa cells. HeLa-Cells were transfected    with RNActive encoding luciferase, which has been complexed with    different ratios of nona-arginine or Poly-L-Arginine, respectively.    24 h later luciferase-activity was measured. Apparently, a mass    ration of less than 2:1 (RNA:nona-arginine) appears to be    advantageous (see FIG. 8).-   b) In comparison, complexation with (high molecular mass)    poly-L-arginine does not increase luciferase-activity at a    significant level. Thus, (high molecular mass) poly-L-arginine does    not appear to be suitable for transfection of mRNA (see FIG. 8).

Example 7—Luciferase Expression Upon Transfection of Complexes of RNAwith Hepta-Arginine ((Arg)_(z)) in HeLa Cells (Comparative Example)

HeLa-Cells were transfected with RNActive encoding luciferase, which hasbeen complexed with different ratios of hepta-arginine ((Arg)₇). 24 hlater luciferase-activity was measured. Apparently, complexation withhepta-arginine ((Arg)₇) does not increase luciferase-activity at asignificant level. Thus, hepta-arginine ((Arg)₇) does not appear to besuitable for transfection of mRNA (see FIG. 9).

Example 8—Immune Stimulation Upon Transfection of Complexes of RNA withHepta-Arginine ((Arg)₇) (Comparative Example) a) TransfectionExperiments

-   -   Transfection Experiments were carried out for hepta-arginine        ((Arg)₇) analogously to the experiments in Example 5 as shown        above.        b) Results of Immunostimulatory Effect of RNA Complexed with        Hepta-Arginine ((Arg)₇)    -   i) HPBMC cells were incubated with RNA complexed with        hepta-arginine ((Arg)₇) for 24 h as disclosed above, wherein the        mass ratio of RNA:(Arg)₇ was 1:1. Then, IL-6 production was        measured in the cell supernatants using ELISA. As a result,        HPBMC cells showed a significant IL-6 production, i.e. a        significant immunostimulatory effect of RNA complexed with        hepta-arginine ((Arg)₇) (see FIG. 10).    -   ii) HPBMC cells were furthermore incubated with RNA complexed        with hepta-arginine ((Arg)₇) for 24 h as disclosed above,        wherein the mass ratio of RNA:(Arg)₇ was 1:1. Then, THF-alpha        production was measured in the cell supernatants using ELISA. As        a result, HPBMC cells also showed a significant TNF-alpha        production, i.e. a significant immunostimulatory effect of RNA        complexed with hepta-arginine ((Arg)₇) (see FIG. 11).

Example 9—Determination of the Effect of Histidin on the TransfectionEfficiency

To determine the effect of Histidin on the transfection efficiency atransfection was carried out analogously to the transfection experimentsabove using peptides with different Histidine content. Therefore, 4 μgstabilized luciferase mRNA according to SEQ ID NO: 36 (Luc-RNActive)were mixed in molar ratios with the respectively peptide (according toformula I), particularly R9, R9H3 or H3R9H3, thereby forming a complex.Afterwards the resulting solution was adjusted with water to a finalvolume of 50 μl und incubated for 30 minutes at room temperature. Theused ratios are in each experiment 1:10000, 1:5000 and 1:1000.HeLa-cells (150×10³/well) were then seeded 1 day prior to transfectionon 24-well microtiter plates leading to a 70% confluence whentransfection was carried out. For transfection 50 μl of theRNA/(peptide)-solution were mixed with 250 μl serum free medium andadded to the cells (final RNA concentration: 13 μg/ml). Prior toaddition of the transfection solution the HeLa-cells were washed gentlyand carefully 2 times with 1 ml Optimen (Invitrogen) per well. Then, thetransfection solution (300 μl per well) was added to the cells and thecells were incubated for 4 h at 37° C. Subsequently 300 μl RPMI-medium(Camprex) containing 10% FCS was added per well and the cells wereincubated for additional 20 h at 37° C. The transfection solution wassucked off 24 h after transfection and the cells were lysed in 300 μllysis buffer (25 mM Tris-PO₄, 2 mM EDTA, 10% glycerol, 1% Triton-X 100,2 mM DTT). The supernatants were then mixed with luciferin buffer (25 mMGlycylglycin, 15 mM MgSO₄, 5 mM ATP, 62.5 μM luciferin) and luminiscencewas detected using a luminometer (Lumat LB 9507 (Berthold Technologies,Bad Wildbad, Germany)).

The results are shown in FIG. 19. As can be seen, a stetch of 3histidines at one terminal end already increases the transfectionefficacy of the complexed RNA, wherein a stetch of 3 histidines at bothterminal ends significantly increases the transfection efficacy of thecomplexed RNA.

Example 10—Determination of the Effect of Neutral Amino Acids on theTransfection Efficiency

To determine the effect of neutral amino acids on the transfectionefficiency a further transfection experiment was carried out analogouslyto the transfection experiments above in Example 9 using the peptideH3R9CCS. The results of this additional experiment are shown in FIG. 20.

Example 11—Immunostimulation Using R9H3 in hPBMCs

The effect of R9H3 on immunostimulation was tested in hPBMCs. Therefore,a complex of R9H3 and RNA as shown above in Example 3 was prepared.Furthermore, HPBMC cells from peripheral blood of healthy donors wereisolated using a Ficoll gradient and washed subsequently with 1×PBS(phophate-buffered saline). The cells were then seeded on 96-wellmicrotiter plates (200×10′/well). The hPBMC cells were incubated for 24h, as described under Example 4, supra, with 10 μl of the RNA/peptidecomplex (RNA final concentration: 6 μg/ml; the same amounts of RNA wereused) in X-VIVO 15 Medium (BioWhittaker). The immunostimulatory effectupon the hPBMC cells was measured by detecting the cytokine production(Interleukin-6 und Tumor necrose factor alpha).

Therefore, ELISA microtiter plates (Nunc Maxisorb) were incubated overnight (o/n) with binding buffer (0.02% NaN3, 15 mM Na2CO3, 15 mM NaHCO3,pH 9.7), additionally containing a specific cytokine antibody. Cellswere then blocked with 1×PBS, containing 1% BSA (bovine serum albumin).The cell supernatant was added and incubated for 4 h at 37° C.Subsequently, the microtiter plate was washed with 1×PBS, 0.05% Tween-20and then incubated with a Biotin-labelled secondary antibody (BDPharmingen, Heidelberg, Germany). Streptavidin-coupled horseraddishperoxidase was added to the plate. Then, the plate was again washed with1×PBS, containing 0.05% Tween-20, and ABTS(2,2′-azino-bis(3-ethyl-benzthiazoline-6-sulfonic acid) was added as asubstrate. The amount of cytokine was determined by measuring theabsorption at 405 nm (OD405) using a standard curve with recombinantCytokines (BD Pharmingen, Heidelberg, Germany) with the SunriseELISA-Reader from Tecan (Crailsheim, Germany). The results are seen inFIGS. 21 and 22. As can be seen, a significant immunostimulation wasexhibited at a ratio of 1:5000 RNA:R9H3.

1. A method for treating a subject having hemophilia B comprisingadministering a pharmaceutical composition comprising an effectiveamount of a mRNA encoding PTC (Factor IX).
 2. The method of claim 1,wherein the pharmaceutical composition comprises a lipid.
 3. The methodof claim 2, wherein the lipid comprises a cationic lipid.
 4. The methodof claim 3, wherein the lipid comprises DOTMA, DOPE, DOSPA, DOTAPDC-Chol or EDMPC.
 5. The method of claim 1, the mRNA encoding PTCcomprises a polypeptide coding sequence having a G/C content that isincreased relative to the G/C content of the wild type sequence for PTC.6. The method of claim 1, wherein the mRNA comprises a 5′ cap structure.7. The method of claim 1, wherein the mRNA additionally comprises apoly-A tail of 10 to 200 adenosine nucleotides.
 8. The method of claim1, wherein the mRNA further comprises a 5′ and/or a 3′ untranslatedregion (UTR).
 9. The method of claim 1, wherein the mRNA comprises afurther chemical modification relative to a naturally occurring mRNA.10. The method claim 9, wherein the mRNA comprises at least one furthernucleotide that is substituted with a nucleotide analog selected fromthe group consisting of: 2′-deoxy-2′-fluoro-oligoribonucleotide(2′-fluoro-2′-deoxycytidine-5′-triphosphate,2′-fluoro-2′-deoxyuridine-5′-triphosphate), 2′-deoxy-2′-deamineoligoribonucleotide (2′-amino-2′-deoxycytidine-5′-triphosphate,2′-amino-2′-deoxyuridine-5′-triphosphate), 2′-O-alkyloligoribonucleotide, 2′-deoxy-2′-C-alkyl oligoribonucleotide(2′-O-methylcytidine-5′-triphosphate, 2′-methyluridine-5′-triphosphate),2′-C-alkyl oligoribonucleotide, and isomers thereof(2′-aracytidine-5′-triphosphate, 2′-arauridine-5′-triphosphate), orazidotriphosphate (2′-azido-2′-deoxycytidine-5′-triphosphate,2′-azido-2′-deoxyuridine-5′-triphosphate)4-thio-uridine-5′-(mono)phosphate,2-Aminopurine-riboside-5′-(mono)phosphate,5-Aminoallylcytidine-5′-(mono)phosphate,5-Aminoallyluridine-5′-(mono)phosphate,5-Bromocytidine-5′-(mono)phosphate,5-Bromo-2′-deoxycytidine-5′-(mono)phosphate,5-Bromouridine-5′-(mono)phosphate,5-Bromo-2′-deoxyuridine-5′-(mono)phosphate,5-Iodocytidine-5′-(mono)phosphate,5-Iodo-2′-deoxycytidine-5′-(mono)phosphate,5-Iodouridine-5′-(mono)phosphate,5-Iodo-2′-deoxyuridine-5′-(mono)phosphate,5-Propynyl-2′-deoxycytidine-5′-(mono)phosphate,5-Propynyl-2′-deoxyuridine-5′-(mono)phosphate,5-formylcytidine-5′-(mono)phosphate,5,2′-O-dimethylcytidine-5′-(mono)phosphate,5-hydroxymethylcytidine-5′-(mono)phosphate,5-formyl-2′-O-methylcytidine-5′-(mono)phosphate,5,2′-O-dimethyluridine-5′-(mono)phosphate,5-methyl-2-thiouridine-5′-(mono)phosphate,5-hydroxyuridine-5′-(mono)phosphate,5-methoxyuridine-5′-(mono)phosphate, uridine 5-oxyaceticacid-5′-(mono)phosphate, uridine 5-oxyacetic acid methylester-5′-(mono)phosphate,5-(carboxyhydroxymethyl)uridine-5′-(mono)phosphate,5-(carboxyhydroxymethyl)uridine methyl ester-5′-(mono)phosphate,5-methoxycarbonylmethyluridine-5′-(mono)phosphate,5-methoxycarbonylmethyl-2′-O-methyluridine-5′-(mono)phosphate,5-methoxycarbonylmethyl-2-thiouridine-5′-(mono)phosphate,5-aminomethyl-2-thiouridine-5′-(mono)phosphate,5-methylaminomethyluridine-5′-(mono)phosphate,5-methylaminomethyl-2-thiouridine-5′-(mono)phosphate,5-methylaminomethyl-2-selenouridine-5′-(mono)phosphate,5-carbamoylmethyluridine-5′-(mono)phosphate,5-carbamoylmethyl-2′-O-methyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyl-2′-O-methyluridine-5′-(mono)phosphate,5-carboxymethylaminomethyl-2-thiouridine-5′-(mono)phosphate,5-carboxymethyluridine-5′-(mono)phosphate,5-methyldihydrouridine-5′-(mono)phosphate,5-taurinomethyluridine-5′-(mono)phosphate,5-taurinomethyl-2-thiouridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)uridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)-2-thiouridine-5′-(mono)phosphate,5-(isopentenylaminomethyl)-2′-O-methyluridine-5′-(mono)phosphate,6-Azacytidine-5′-(mono)phosphate, 7-Deazaadenosine-5′-(mono)phosphate,7-Deazaguanosine-5′-(mono)phosphate, 8-Azaadenosine-5′-(mono)phosphate,8-Azidoadenosine-5′-(mono)phosphate, Pseudouridine-5′-(mono)phosphate,2′-Amino-2′-deoxycytidine-(mono)phosphate,2′-Fluorothymidine-5′-(mono)phosphate, inosine-5′-(mono)phosphate, and2′-O-Methyl-inosine-5′-(mono)phosphate.
 11. The method of claim 10,wherein the mRNA comprises at least one nucleotide that is substitutedwith a nucleotide analog selected from the group consisting of:2-amino-6-chloropurineriboside-5′-triphosphate,2-aminoadenosine-5′-triphosphate, 2-thiocytidine-5′-triphosphate,2-thiouridine-5′-triphosphate, 4-thiouridine-5′-triphosphate,5-aminoallylcytidine-5′-triphosphate,5-aminoallyluridine-5′-triphosphate, 5-bromocytidine-5′-triphosphate,5-bromouridine-5′-triphosphate, 5-iodocytidine-5′-triphosphate,5-iodouridine-5′-triphosphate, 5-methylcytidine-5′-triphosphate,5-methyluridine-5′-triphosphate, 6-azacytidine-5′-triphosphate,6-azauridine-5′-triphosphate, 6-chloropurineriboside-5′-triphosphate,7-deazaadenosine-5′-triphosphate, 7-deazaguanosine-5′-triphosphate,8-azaadenosine-5′-triphosphate, 8-azidoadenosine-5′-triphosphate,benzimidazole-riboside-5′-triphosphate,N1-methyladenosine-5′-triphosphate, N1-methylguanosine-5′-triphosphate,N6-methyladenosine-5′-triphosphate, O6-methylguanosine-5′-triphosphate,pseudouridine-5′-triphosphate, or puromycin-5′-triphosphate,xanthosine-5′-triphosphate.
 12. The method of claim 11, wherein thenucleotide analog is chosen from the group consisting of:5-methylcytidine 5′-triphosphate and pseudouridine 5′-triphosphate. 13.The method of claim 12, wherein the nucleotide analog is pseudouridine5′-triphosphate.
 14. The method of claim 1, wherein the pharmaceuticalcomposition comprises water, a saline solution or Ringer-Lactatsolution.
 15. The method of claim 1, wherein the pharmaceuticalcomposition comprises a pH of 7.0 to 7.6.
 16. The method of claim 1,wherein the pharmaceutical composition is administered by injection. 17.The method of claim 16, wherein the pharmaceutical composition isadminister by intramuscular or intravenous injection.